sİtoplazmİk zar ve sİtoplazma -...

SİTOPLAZMİK ZAR ve SİTOPLAZMA

Sitoplazmik Zarın İçeriği ve Fonksiyonu • Hem gram negatif hem gram pozitif bakterilerde sitoplazmayı saran ve sitoplazma ile hücre çeperi arasında yer alan bu zarın ayrı bir yapı olduğu Knaysi (1938) tarafından gösterilmiştir. • İki tabakalı fosfolipit yapıda olan zar boyanarak yada hücreler lizozimle eritildikten sonra santrifüj edilip ayırılarak gösterilebilir.

Yrd.Doç.Dr.Yosun MATER (Brock Biology of Microorganisms 13th Edition 2012)

CHAPTER 3 • Cell Structure and Function in Bacteria and Archaea 51

in diameter are marginal. Thus, structures occasionally observedin nature of 0.1 !m or smaller that “look” like bacterial cells arealmost certainly not so. Despite this, many very small prokary-otic cells are known and many have been grown in the laboratory.The open oceans, for example, contain 104–105 prokaryotic cellsper milliliter, and these tend to be very small cells, 0.2–0.4 !m indiameter. We will see later that many pathogenic bacteria are alsovery small. When the genomes of these pathogens are examined,they are found to be highly streamlined and missing many geneswhose functions are supplied to them by their hosts.

MiniQuiz• What physical property of cells increases as cells become

smaller?• How can the small size and haploid genetics of prokaryotes

accelerate their evolution?

II The Cytoplasmic Membraneand Transport

We now consider the structure and function of a critical cellcomponent, the cytoplasmic membrane. The cytoplasmic

membrane plays many roles, chief among them as the “gate-keeper” for substances that enter and exit the cell.

3.3 The Cytoplasmic MembraneThe cytoplasmic membrane is a thin barrier that surrounds thecell and separates the cytoplasm from the cell’s environment. Ifthe membrane is broken, the integrity of the cell is destroyed, thecytoplasm leaks into the environment, and the cell dies. We willsee that the cytoplasmic membrane confers little protection fromosmotic lysis but is ideal as a selective permeability barrier.

Composition of MembranesThe general structure of the cytoplasmic membrane is a phos-pholipid bilayer. Phospholipids contain both hydrophobic (fattyacid) and hydrophilic (glycerol–phosphate) components and canbe of many different chemical forms as a result of variation in thegroups attached to the glycerol backbone (Figure 3.4) As phos-pholipids aggregate in an aqueous solution, they naturally formbilayer structures. In a phospholipid membrane, the fatty acidspoint inward toward each other to form a hydrophobic environ-ment, and the hydrophilic portions remain exposed to the exter-nal environment or the cytoplasm (Figure 3.4b).

The cell’s cytoplasmic membrane, which is 6–8 nanometerswide, can be seen with the electron microscope, where it appearsas two dark-colored lines separated by a lighter area (Figure 3.4c).This unit membrane, as it is called (because each phospholipidleaf forms half of the “unit”), consists of a phospholipid bilayerwith proteins embedded in it (Figure 3.5). Although in a diagramthe cytoplasmic membrane may appear rather rigid, in reality it issomewhat fluid, having a consistency approximating that of alow-viscosity oil. Some freedom of movement of proteins withinthe membrane is possible, although it remains unclear exactly

how extensive this is. The cytoplasmic membranes of someBacteria are strengthened by molecules called hopanoids. Thesesomewhat rigid planar molecules are structural analogs ofsterols, compounds that strengthen the membranes of eukaryoticcells, many of which lack a cell wall.

Membrane ProteinsThe major proteins of the cytoplasmic membrane havehydrophobic surfaces in their regions that span the membraneand hydrophilic surfaces in their regions that contact the envi-ronment and the cytoplasm (Figures 3.4 and 3.5). The outer sur-face of the cytoplasmic membrane faces the environment and ingram-negative bacteria interacts with a variety of proteins thatbind substrates or process large molecules for transport into thecell (periplasmic proteins, see Section 3.7). The inner side of thecytoplasmic membrane faces the cytoplasm and interacts withproteins involved in energy-yielding reactions and other impor-tant cellular functions.

Many membrane proteins are firmly embedded in the mem-brane and are called integral membrane proteins. Other proteinshave one portion anchored in the membrane and extramem-brane regions that point into or out of the cell (Figure 3.5). Still

UN

IT 1

Figure 3.4 Phospholipid bilayer membrane. (a) Structure of thephospholipid phosphatidylethanolamine. (b) General architecture of abilayer membrane; the blue balls depict glycerol with phosphate and (or)other hydrophilic groups. (c) Transmission electron micrograph of a mem-brane. The light inner area is the hydrophobic region of the model mem-brane shown in part b.

Hydrophobicregion

Hydrophilicregion

Glycerol

Hydrophilicregion

Fatty acids

(b)

(c)

Glycerophosphates

Fatty acids

(a)

C O

O

C OO

C

C

H

H

H

PO

O

CHH

–O

O

CH2

CH2+NH3

Ethanolamine

Phosphate

Fatty acids

H3C

H3C

G. W

agne

r

Yrd.Doç.Dr.Yosun MATER

(Brock Biology of Microorganisms 13th Edition 2012)

Figure 3.6 General structure of lipids. (a) The ester linkage and (b) the ether linkage. (c) Isoprene, the parent structure of the hydropho-bic side chains of archaeal lipids. By contrast, in lipids of Bacteria andEukarya, the side chains are composed of fatty acids (see Figure 3.4a).

UNIT 1 • Basic Principles of Microbiology52

other proteins, called peripheral membrane proteins, are notmembrane-embedded but nevertheless remain firmly associatedwith membrane surfaces. Some of these peripheral membraneproteins are lipoproteins, molecules that contain a lipid tail thatanchors the protein into the membrane. Peripheral membrane pro-teins typically interact with integral membrane proteins in impor-tant cellular processes such as energy metabolism and transport.

Proteins in the cytoplasmic membrane are arranged in clusters(Figure 3.5), a strategy that allows proteins that need to interactto be adjacent to one another. The overall protein content of themembrane is quite high, and it is thought that the variation inlipid bilayer thickness (6–8 nm) is necessary to accommodatethicker and thinner patches of membrane proteins.

Archaeal MembranesIn contrast to the lipids of Bacteria and Eukarya in which esterlinkages bond the fatty acids to glycerol, the lipids of Archaeacontain ether bonds between glycerol and their hydrophobic sidechains (Figure 3.6). Archaeal lipids lack true fatty acid side chainsand instead, the side chains are composed of repeating units ofthe hydrophobic five-carbon hydrocarbon isoprene (Figure 3.6c).

The cytoplasmic membrane of Archaea can be constructed ofeither glycerol diethers (Figure 3.7a), which have 20-carbon sidechains (the 20-C unit is called a phytanyl group), or diglyceroltetraethers (Figure 3.7b), which have 40-carbon side chains. Inthe tetraether lipid, the ends of the phytanyl side chains that

point inward from each glycerol molecule are covalently linked.This forms a lipid monolayer instead of a lipid bilayer membrane(Figure 3.7d, e). In contrast to lipid bilayers, lipid monolayermembranes are extremely resistant to heat denaturation and aretherefore widely distributed in hyperthermophiles, prokaryotesthat grow best at temperatures above 808C. Membranes with amixture of bilayer and monolayer character are also possible,with some of the inwardly opposing hydrophobic groups cova-lently bonded while others are not.

Figure 3.5 Structure of the cytoplasmic membrane. The inner surface (In) faces the cytoplasm and theouter surface (Out) faces the environment. Phospholipids compose the matrix of the cytoplasmic membranewith proteins embedded or surface associated. Although there are some chemical differences, the overallstructure of the cytoplasmic membrane shown is similar in both prokaryotes and eukaryotes (but an excep-tion to the bilayer design is shown in Figure 3.7e).

Integralmembraneproteins Phospholipid

molecule

Phospholipids

Hydrophilicgroups

Hydrophobicgroups

Out

In

6–8 nm

O–P

O–

BacteriaEukarya

(a)

CH2 H2C C C

CH3

H

Archaea

(b) (c)

O–H2C

H2C O C

O

R

O C

O

R

O P

O

O–

H2C

H2C O C R

O C R

O

OHC HC

EsterEther


Besin maddelerinin taşınması:

• En önemli özelliği “yarı-geçirgen” (=semi-permeable) olmasıdır. Bu özelliği ile hem ozmotik bir baraj hem bir köprü olarak iş görür. Çevrede yer alan çoğu molekül sitoplazmik zarda yer alan lipit tabakaları doğrudan geçemezler. Taşıma proteinleri denilen ve çift tabakalı fosfolipit tabakayı boydan boya geçen proteinler yardımıyla şeker ve diğer besin maddeleri sitoplazmaya geçerler; besin ve enerji kaynağı olarak kullanılırlar.

• Bakteriler zaman zaman zararlı maddelerinde geçişine izin verebilirler. Bunun sebebi taşıma proteinlerinin bir veya birkaç bileşeni özellikle tanımasıdır.

• Genellikle bakteri içindeki madde yoğunlu canlının bulunduğu çevredeki madde yoğunluğundan fazladır bu nedenle hücre içine madde alınımında enerji harcanır.


Enerji mekanizması ve elektron taşıma sistemi:

• Bakterilerde hücre zarı, hücrenin enerji mekanizmasında önemli bir role sahiptir.

• Sitoplazmik zarda yer alan enzimler ve diğer moleküller proton pompaları yardımıyla çalışırlar. Bu proteinler ve bu sistemde yer alan diğer moleküller elektron taşıma sistemi adını alır. Çünkü elektronlar protonlardan ayrılır ve sitoplazmik zarın dış yüzeyinde birikirler. Karmaşık protein yapılı terminal elektron yakalayıcıları ATP sentaz’lar bu elektronları yakalar ve yeniden hücre içine alır. ATP hücrenin temel enerji kaynağıdır. Bu enerji kirpik motorlarının çalıştırılması ve dışarıdan hücre içine madde alınmasında kullanılır.

• Bu zar ökaryotlarda mitokondri zarlarına benzer bir görev yapmaktadır ve suya ilgisi azdır.


• Bakteri hücre çeperi ve hücre zarının kuru ağırlıktaki oranı %20 kadardır. Bunun nedeni bakterilerin iç basınçlarının çok yüksek olmasıdır. İç basınçları %10-20’lik sakaroz çözeltisine eşittir.

• Bu nedenle ancak sağlam bir hücre zarı ve hücre çeperi yardımıyla kendilerini patlamaktan koruyabilirler. Hücre çeperleri oluşturması engellenen bakteriler genellikle küre biçiminde hücrelere yani protoplastlara dönüşürler.

• Eğer bakteri hücrelerinin etrafında hücre çeperi kalıntıları bulunuyorsa bu durumda bakterilere sferoblast adı verilir. Bunların etraflarında hücre zarları vardır.


Sitoplazmik zar ve peptidoglikan sentezi: • Sitoplazmik zarda yer alan proteinler, peptidoglikan sentezi ve devrinde de rol oynarlar. Bakterilere uzama ve bölünme emri geldiğinde, peptidoglikanda kırılma meydana gelip yeniden uzatılarak yapılandırılır. • Sitoplazmik zar proteinleri bu kırılma ve yeniden yapılanma aşamasında yardım ederler.

• Ayrıca antibiyotik bağlayan proteinlerde burada yer alır. LTA ve LPS sitoplazmik zara bağlıdır.


Salınım sistemi: • Sitoplazmik zarda yer alan proteinler salgılanmış proteinlerdir. Sitoplazmada yapılırlar ve periplazma içine salınırlar. Bu karmaşık proteinlerden oluşan sisteme salgı sistemi adı verilir.

• Bakteriler ürettikleri bütün proteinleri salgılamak istemezler. Bazılarını periplazmada tutarlar, bazılarını dış çevreye salarlar.

• Bu tutulan proteinleri amino uçlarında yer alan kısa amino asit sırasının varlığına bakarak belirleyebiliriz.

• Bu kısa parça kesilip uzaklaştırıldığında protein salınır.


Düzenleyici proteinler: • Bakterilerin sitoplazmik zarı kısmen beyin gibi de görev alır.

• Çünkü burada yer alan bazı duyusal proteinler hücrenin dışındaki çevre şartları ile ilgili değişiklikleri hücre içine iletirler.

• Bu şekilde hücre dışarıda algıladığı durumlara bağlı olarak hücre içinde ve dışında uygun düzenlemeler yapılır. Ör: insan sindirim sistemine giren bir bakteri ortama hemen tutunması gerekir bu nedenle hemen pili üretir ve o bölgeye tutunur.

• Bu düzenleyici proteinler yalnız sitoplazma zarında yer almaz hücre içinde de yer alır. Bu düzenlemelerin tam anlamıyla yapılabilmesi için hem sitoplazma zarı hem de hücre içinde yer alan düzenleyici proteinlerin birlikte çalışması gerekir.


SİTOPLAZMA • Bakteri hücrelerinin sitoplazmasında yer alan enzimler yardımıyla glukoz ve diğer karbon kaynakları okside edilerek doğrudan ATP elde edilir.

• Bu enzimlerden bazıları sitoplazmik zarda yer alan elektron taşıma sistemi yardımıyla dolaylı olarak ATP elde edilmesinde görev alır.

• Buna ek olarak zaman zaman bakterilerde yer alan hücre içinde zar katlanmaları şeklinde gözlenen ve mezozom adını alan, hem enerji eldesin de hem de gaz alış verişinde görev aldığı düşünülen yapılardır. Fotosentetik bakterilerde fotosentezden sorumlu pigmentler vardır. Bunlarda yine mezozomların üzerinde yer alırlar.

• Peptidoglikan tabakanın alt birimleri sitoplazmada hazırlanır ve son birleştirme aşamasında periplazmik alana geçerler. Yine bazı yedek maddeler (Ör: poly- β hidroksibütirik asit granülleri) sitoplazma içinde granüller halinde depo edilebilirler.


Ribozomlar: • Sitoplazmada serbest halde yer alırlar. Mezozom benzeri zar katlanmalarına tutunmuş olarak ta görülebilirler.

• Bakteri hücrelerinde yer alan RNA’nın %80’den fazlası ribozomlarda yer alır.

• Prokaryotlardaki ribozomlar hemen hemen küresel yapıda görünürler. 200Å çapında, 70S çökelme sabitine sahiptirler. Alt birimleri 50S ve 30S’ten oluşur. Bakteri genetik materyali: • Hücrenin genetik bilgilerini içeren DNA (=Nukleoid) ve onunla bağlantı halinde bulunan proteinlerde sitoplazmada yer alır.

• E.coli bakterisi DNA’sı yaklaşık 1 mm boyunda olduğu ve 3 µm boyundaki bakterinin içine bu DNA’nın sığması için sıkıca paketlendiği belirlenmiştir.


UNIT 1 • Principles of Microbiology32

(b) Eukaryote

(a) Prokaryote

Cytoplasm Nucleoid

Cell wall Cytoplasmicmembrane

Plasmid

Cytoplasmicmembrane

Endoplasmicreticulum

membrane

Ribosomes

Ribosomes

Nucleus

Nucleolus

Cytoplasm

Golgicomplex

10 µm

0.5 µm

Nuclear

Mitochondrion

Chloroplast

Figure 2.11 Internal structure of cells. Note differences in scale andinternal structure between the prokaryotic and eukaryotic cells.

Ribosomes interact with cytoplasmic proteins and messenger andtransfer RNAs in the key process of protein synthesis (translation).

The cell wall lends structural strength to a cell. The cell wall isrelatively permeable and located outside the membrane (Figure2.11a); it is a much stronger layer than the membrane itself. Plantcells and most microorganisms have cell walls, whereas animalcells, with rare exceptions, do not.

Prokaryotic and Eukaryotic CellsExamination of the internal structure of cells reveals two distinctpatterns: prokaryote and eukaryote (Figure 2.12). Eukaryoteshouse their DNA in a membrane-enclosed nucleus and are typi-cally much larger and structurally more complex than prokaryoticcells. In eukaryotic cells the key processes of DNA replication, tran-scription, and translation are partitioned; replication and transcrip-tion (RNA synthesis) occur in the nucleus while translation (proteinsynthesis) occurs in the cytoplasm. Eukaryotic microorganismsinclude algae and protozoa, collectively called protists, and the fungiand slime molds. The cells of plants and animals are also eukaryoticcells. We consider microbial eukaryotes in detail in Chapter 20.

A major property of eukaryotic cells is the presence of mem-brane-enclosed structures in the cytoplasm called organelles.These include, first and foremost, the nucleus, but also mitochon-dria and chloroplasts (the latter in photosynthetic cells only)(Figures 2.2a and 2.12c). As mentioned, the nucleus houses the cell’sgenome and is also the site of RNA synthesis in eukaryotic cells.Mitochondria and chloroplasts are dedicated to energy conserva-tion and carry out respiration and photosynthesis, respectively.

In contrast to eukaryotic cells, prokaryotic cells have a simplerinternal structure in which organelles are absent (Figures 2.11a

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D. B

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(c) Eukarya

Cytoplasmicmembrane

Nucleus

Cellwall

Mitochondrion

Prokaryotes Eukaryote

Figure 2.12 Electron micrographs of sectioned cells from each of the domains of living organisms.(a) Heliobacterium modesticaldum; the cell measures 1 * 3 !m. (b) Methanopyrus kandleri; the cell measures0.5 * 4 !m. Reinhard Rachel and Karl O. Stetter, 1981. Archives of Microbiology 128:288–293. © Springer-Verlag GmbH & Co. KG. (c) Saccharomyces cerevisiae; the cell measures 8 !m in diameter.


UNIT 3 • Molecular Biology and Gene Expression158

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mal

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20

30

40

50

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70

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lac operon(lactosedegradation)

trp operon(tryptophanbiosynthesis)

his operon(histidinebiosynthesis)

Escherichia coli K-12

Not1 restrictionsites, in kbp

Figure 6.10 The chromosome ofEscherichia coli strain K-12. The E. coli chromo-some contains 4,639,221 base pairs and 4288open reading frames (an indicator of genes;Section 6.17). On the outer edge of the map, thelocations of a few genes are indicated. A fewoperons are also shown, with their directions oftranscription. Around the inner edge, the numbersfrom 0 to 100 refer to map position in minutes.Note that 0 is located by convention at the thr

locus. Replication proceeds bidirectionally fromthe origin of DNA replication, oriC, at 84.3 min.The inner circle shows the locations, in kilobasepairs, of the sites where the restriction enzymeNotI cuts. The origins and directions of transfer ofa few Hfr strains are also shown (arrows). Thelocations of five copies of the transposable ele-ment IS3 found in a particular strain are shown inblue. The site where bacteriophage lambda inte-grates is shown in red. If lambda were present, it

would add an extra 48.5 kbp (slightly over 1 min)to the map. The genes of the maltose regulon,which includes several operons, are indicated bygreen labels. The maltose genes are abbreviatedmal except for lamB, which encodes an outermembrane protein for maltose uptake that is alsothe receptor for bacteriophage lambda. The generpsL (73 min) encodes a ribosomal protein. Thisgene was once called str because mutations in itlead to streptomycin resistance.

Arrangement of Genes on the Escherichia coli ChromosomeGenetic mapping of the genes that encode the enzymes of a sin-gle biochemical pathway in E. coli has shown that these genes areoften clustered. On the genetic map in Figure 6.10, a few suchclusters are shown. Notice, for instance, the gal gene cluster at18 min, the trp gene cluster at about 28 min, and the his cluster at

44 min. Each of these gene clusters constitutes an operon that istranscribed as a single mRNA carrying multiple coding sequences,that is, a polycistronic mRNA (Section 6.15).

Genes for some other biochemical pathways in E. coli are notclustered. For example, genes for arginine biosynthesis (arggenes) are scattered throughout the chromosome. The early dis-covery of multigene operons and their use in studying gene


• B a k t e r i D N A ’ s ı nukleoid bölgede iki şekilde, katlanmış ve katlanmamış (açık) olarak bulunurlar.

• DNA’nın çok sıkı bir şekilde kendi üzerine k a t l a n d ı ğ ı ş e k i l s ü p e r s a r m a l (=supercoil) adını alır.

CHAPTER 6 • Molecular Biology of Bacteria 155

UN

IT 3

MiniQuiz• What does antiparallel mean in terms of the structure of double-

stranded DNA?• Define the term complementary when used to refer to two

strands of DNA.• Define the terms denaturation, reannealing, and hybridization as

they apply to nucleic acids.• Why do GC-rich molecules of DNA melt at higher temperatures

than AT-rich molecules?

6.3 SupercoilingIf linearized, the Escherichia coli chromosome would be over 1 mm in length, about 700 times longer than the E. coli cell itself.How is it possible to pack so much DNA into such a little space?The solution is the imposition of a “higher-order” structure onthe DNA, in which the double-stranded DNA is further twistedin a process called supercoiling. Figure 6.8 shows how supercoil-ing occurs in a circular DNA duplex. If a circular DNA moleculeis linearized, any supercoiling is lost and the DNA becomes“relaxed.” When relaxed, a DNA molecule has exactly the num-ber of turns of the helix predicted from the number of base pairs.

Supercoiling puts the DNA molecule under torsion, much likethe added tension to a rubber band that occurs when it is twisted.DNA can be supercoiled in either a positive or a negative man-ner. In positive supercoiling the double helix is overwound,whereas in negative supercoiling the double helix is underwound.Negative supercoiling results when the DNA is twisted about its

1.2

1.0

A26

0

0.8

76 80 84 88 92 9672

°C

Melting

Single strands

Double strand

Tm= 85.0°

GT

T A

AC

G C

C

G C

C

TA

A

A

TA A

T

T

A T

T

CG

G

C

G

C

T

A

T

A

A

T

A

T

C

G

G

G

Figure 6.7 Thermal denaturation of DNA. DNA absorbs more ultravi-olet radiation at 260 nm as the double helix is denatured. The transition is quite abrupt, and the temperature of the midpoint, Tm, is proportional to the GC content of the DNA. Although the denatured DNA can berenatured by slow cooling, the process does not follow a similar curve.Renaturation becomes progressively more complete at temperatures wellbelow the Tm and then only after a considerable incubation time.

Nick

Relaxed, nicked circular DNA(b)

Supercoiled circular DNA(c)

Chromosomal DNA with supercoiled domains(d)

Relaxed, covalently closed circular DNA(a)

Proteins

Supercoileddomain

Seal

Rotate one end of broken strand around helix and seal

Break one strand

Break one strand

Figure 6.8 Supercoiled DNA. (a–c) Relaxed, nicked, and supercoiledcircular DNA. A nick is a break in a phosphodiester bond of one strand. (d) In fact, the double-stranded DNA in the bacterial chromosome isarranged not in one supercoil but in several supercoiled domains, asshown here.

Yrd.Doç.Dr.Yosun MATER (Brock Biology of

Microorganisms 13th Edition 2012)


UN

IT 3

MiniQuiz• What does antiparallel mean in terms of the structure of double-

stranded DNA?• Define the term complementary when used to refer to two

strands of DNA.• Define the terms denaturation, reannealing, and hybridization as

they apply to nucleic acids.• Why do GC-rich molecules of DNA melt at higher temperatures

than AT-rich molecules?

6.3 SupercoilingIf linearized, the Escherichia coli chromosome would be over 1 mm in length, about 700 times longer than the E. coli cell itself.How is it possible to pack so much DNA into such a little space?The solution is the imposition of a “higher-order” structure onthe DNA, in which the double-stranded DNA is further twistedin a process called supercoiling. Figure 6.8 shows how supercoil-ing occurs in a circular DNA duplex. If a circular DNA moleculeis linearized, any supercoiling is lost and the DNA becomes“relaxed.” When relaxed, a DNA molecule has exactly the num-ber of turns of the helix predicted from the number of base pairs.

Supercoiling puts the DNA molecule under torsion, much likethe added tension to a rubber band that occurs when it is twisted.DNA can be supercoiled in either a positive or a negative man-ner. In positive supercoiling the double helix is overwound,whereas in negative supercoiling the double helix is underwound.Negative supercoiling results when the DNA is twisted about its

1.2

1.0

A26

0

0.8

76 80 84 88 92 9672

°C

Melting

Single strands

Double strand

Tm= 85.0°

GT

T A

AC

G C

C

G C

C

TA

A

A

TA A

T

T

A T

T

CG

G

C

G

C

T

A

T

A

A

T

A

T

C

G

G

G

Figure 6.7 Thermal denaturation of DNA. DNA absorbs more ultravi-olet radiation at 260 nm as the double helix is denatured. The transition is quite abrupt, and the temperature of the midpoint, Tm, is proportional to the GC content of the DNA. Although the denatured DNA can berenatured by slow cooling, the process does not follow a similar curve.Renaturation becomes progressively more complete at temperatures wellbelow the Tm and then only after a considerable incubation time.

Nick

Relaxed, nicked circular DNA(b)

Supercoiled circular DNA(c)

Chromosomal DNA with supercoiled domains(d)

Relaxed, covalently closed circular DNA(a)

Proteins

Supercoileddomain

Seal

Rotate one end of broken strand around helix and seal

Break one strand

Break one strand

Figure 6.8 Supercoiled DNA. (a–c) Relaxed, nicked, and supercoiledcircular DNA. A nick is a break in a phosphodiester bond of one strand. (d) In fact, the double-stranded DNA in the bacterial chromosome isarranged not in one supercoil but in several supercoiled domains, asshown here.

• Bu tip katlanma bakteriler adına oldukça önemlidir. Bazen bakterilerde histon benzeri proteinler yer alır ve DNA’ya tutunurlar ve D N A ’ n ı n b u y a p ı y ı k o r u m a s ı n a y a r d ı m ederler.

• DNA sentezinde yer alan e n z i m l e r v e R N A’ d a sitoplazmada yer alır.



Plazmitler: • Birçok bakteri türünde normal bakteri genomundan farklı olarak sitoplazmada yer alan dairesel DNA yapılarına plazmit adı verilir.

• Bunlar toksin yapımı, genetik madde aktarımı, antibiyotiklere direnç, farklı antijenik özellikleri ve daha birçok özelliği taşıyan özel yapılardır.

• Bakteri genetiği çalışmalarında sıklıkla kullanılan çok önemli yapılardır.

UNIT 4 • Virology, Genetics, and Genomics280

(Section 10.13) that allow the plasmid to integrate into the hostchromosome. In addition, the F plasmid has a large region ofDNA, the tra region, containing genes that encode transfer func-tions. Many genes in the tra region are involved in mating pairformation, and most of these have to do with the synthesis of asurface structure, the sex pilus ( Section 3.9). Only donor cellsproduce these pili. Different conjugative plasmids may haveslightly different tra regions, and the pili may vary somewhat instructure. The F plasmid and its relatives encode F pili.

Pili allow specific pairing to take place between the donor andrecipient cells. All conjugation in gram-negative Bacteria isthought to depend on cell pairing brought about by pili. Thepilus makes specific contact with a receptor on the recipient celland then is retracted by disassembling its subunits. This pullsthe two cells together (Figure 10.17). Following this process,donor and recipient cells remain in contact by binding proteinslocated in the outer membrane of each cell. DNA is then trans-ferred from donor to recipient cell through this conjugationjunction.

Mechanism of DNA Transfer During ConjugationDNA synthesis is necessary for DNA transfer by conjugation.This DNA is synthesized not by normal bidirectional replication( Section 6.10), but by rolling circle replication, a mecha-nism also used by some viruses ( Section 9.10) and shown inFigure 10.18. DNA transfer is triggered by cell-to-cell contact, atwhich time one strand of the circular plasmid DNA is nicked andis transferred to the recipient. The nicking enzyme required to

initiate the process, TraI, is encoded by the tra operon of the Fplasmid. This protein also has helicase activity and thus alsounwinds the strand to be transferred. As this transfer occurs,DNA synthesis by the rolling circle mechanism replaces thetransferred strand in the donor, while a complementary DNAstrand is being made in the recipient. Therefore, at the end of theprocess, both donor and recipient possess complete plasmids.For transfer of the F plasmid, if an F-containing donor cell, whichis designated F+, mates with a recipient cell lacking the plasmid,designated F-, the result is two F+ cells (Figure 10.18).

Transfer of plasmid DNA is efficient and rapid; under favor-able conditions virtually every recipient cell that pairs with adonor acquires a plasmid. Transfer of the F plasmid, comprisingapproximately 100 kbp of DNA, takes about 5 minutes. If theplasmid genes can be expressed in the recipient, the recipientitself becomes a donor and can transfer the plasmid to otherrecipients. In this fashion, conjugative plasmids can spread rap-idly among bacterial populations, behaving much like infectiousagents. This is of major ecological significance because a fewplasmid-containing cells introduced into a population of recipi-ents can convert the entire population into plasmid-bearing (andthus donating) cells in a short time.

Plasmids can be lost from a cell by curing. This may happenspontaneously in natural populations when there is no selectionpressure to maintain the plasmid. For example, plasmids confer-ring antibiotic resistance can be lost without affecting cell viabil-ity if there are no antibiotics in the cells’ environment.

MiniQuiz• In conjugation, how are donor and recipient cells brought into

contact with each other?• Explain how rolling circle DNA replication allows both donor and

recipient to end up with a complete copy of plasmids transferredby conjugation.

• Why does F have two different origins of replication?

IS3

tra region

99.2kbp/0

25 kbp75 kbp

50 kbp

Tn1000

IS3

IS2

oriT

oriV

F plasmid

Figure 10.16 Genetic map of the F (fertility) plasmid of Escherichiacoli. The numbers on the interior show the size in kilobase pairs (theexact size is 99,159 bp). The region in dark green at the bottom of themap contains genes primarily responsible for the replication and segre-gation of the F plasmid. The origin of vegetative replication is oriV. Thelight green tra region contains the genes needed for conjugative transfer.The origin of transfer during conjugation is oriT. The arrow indicates thedirection of transfer (the tra region is transferred last). Insertion sequencesare shown in yellow. These may recombine with identical elements on thebacterial chromosome, which leads to integration and the formation ofdifferent Hfr strains.

C. B

rinto

n

Pilus with attachedphage virions

Figure 10.17 Formation of a mating pair. Direct contact between twoconjugating bacteria is first made via a pilus. The cells are then drawntogether to form a mating pair by retraction of the pilus, which is achievedby depolymerization. Certain small phages (F-specific bacteriophages;

Section 21.1) use the sex pilus as a receptor and can be seen hereattached to the pilus.



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mechanism of plasmid transfer is conjugation, a functionencoded by some plasmids themselves that involves cell-to-cellcontact ( Section 10.9).

Plasmids capable of transferring themselves by cell-to-cellcontact are called conjugative. Not all plasmids are conjugative.Transfer by conjugation is controlled by a set of genes on theplasmid called the tra (for transfer) region. These genes encodeproteins that function in DNA transfer and replication and oth-ers that function in mating pair formation. If a plasmid possess-ing a tra region becomes integrated into the chromosome, theplasmid can then mobilize the chromosomal DNA, which may betransferred from one cell to another ( Section 10.10).

Most conjugative plasmids can only move between closelyrelated species of bacteria. However, some conjugative plasmidsfrom Pseudomonas have a broad host range. This means thatthey are transferable to a wide variety of other gram-negativeBacteria. Such plasmids can transfer genes between distantlyrelated organisms. Conjugative plasmids have been shown totransfer between gram-negative and gram-positive Bacteria,between Bacteria and plant cells, and between Bacteria andfungi. Even if the plasmid cannot replicate independently in thenew host, transfer of the plasmid itself could have important evo-lutionary consequences if genes from the plasmid recombinewith the genome of the new host.

MiniQuiz• How does a plasmid differ from a virus?• How can a large plasmid be differentiated from a small

chromosome?• What function do the tra genes of the F plasmid carry out?

6.7 The Biology of PlasmidsClearly, all plasmids must carry genes that ensure their ownreplication. In addition, some plasmids also carry genes neces-sary for conjugation. Although plasmids do not carry genes thatare essential to the host, plasmids may carry genes that pro-foundly influence host cell physiology. In some cases plasmidsencode properties fundamental to the ecology of the bacterium.For example, the ability of Rhizobium to interact with plants andform nitrogen-fixing root nodules requires certain plasmid func-tions ( Section 25.8). Other plasmids confer special metabolicproperties on bacterial cells, such as the ability to degrade toxicpollutants. Indeed, plasmids are a major mechanism for confer-ring special properties on bacteria and for mobilizing these prop-erties by horizontal gene flow. Some special properties conferredby plasmids are summarized in Table 6.2.

Resistance PlasmidsAmong the most widespread and well-studied groups of plas-mids are the resistance plasmids, usually just called R plasmids,which confer resistance to antibiotics and various other growthinhibitors. Several antibiotic resistance genes can be carried by asingle R plasmid, or, alternatively, a cell may contain several R plas-mids. In either case, the result is multiple resistance. R plasmidswere first discovered in Japan in the 1950s in strains of enteric

bacteria that had acquired resistance to sulfonamide antibiotics.Since then they have been found throughout the world. The emer-gence of bacteria resistant to antibiotics is of considerable medicalsignificance and is correlated with the increasing use of antibioticsfor treating infectious diseases ( Section 26.12). Soon afterthese resistant strains were isolated, it was shown that they couldtransfer resistance to sensitive strains via cell-to-cell contact. Theinfectious nature of conjugative R plasmids permitted their rapidspread through cell populations.

In general, resistance genes encode proteins that either inacti-vate the antibiotic or protect the cell by some other mechanism.Plasmid R100, for example, is a 94.3-kbp plasmid (Figure 6.12)that carries genes encoding resistance to sulfonamides, strepto-mycin, spectinomycin, fusidic acid, chloramphenicol, and tetra-cycline. Plasmid R100 also carries several genes conferringresistance to mercury. Plasmid R100 can be transferred betweenenteric bacteria of the genera Escherichia, Klebsiella, Proteus,Salmonella, and Shigella, but does not transfer to gram-negativebacteria outside the enteric group. Different R plasmids withgenes for resistance to most antibiotics are known. Many drug-resistant modules on R plasmids, such as those on R100, are alsotransposable elements ( Section 12.11), and this, combinedwith the fact that many of these plasmids are conjugative, havemade them a serious threat to traditional antibiotic therapy.

Plasmids Encoding Virulence CharacteristicsPathogenic microorganisms possess a variety of characteristicsthat enable them to colonize hosts and establish infections. Herewe note two major characteristics of the virulence (disease-causing

Table 6.2 Examples of phenotypes conferred by plasmids in prokaryotes

Phenotype class Organismsa

Antibiotic production Streptomyces

Conjugation Wide range of bacteria

Metabolic functionsDegradation of octane, camphor,

naphthalenePseudomonas

Degradation of herbicides AlcaligenesFormation of acetone and butanol ClostridiumLactose, sucrose, citrate, or urea

utilizationEnteric bacteria

Pigment production Erwinia, StaphylococcusGas vesicle production Halobacterium

ResistanceAntibiotic resistance Wide range of bacteriaResistance to toxic metals Wide range of bacteria

VirulenceTumor production in plants AgrobacteriumNodulation and symbiotic nitrogen

fixationRhizobium

Bacteriocin production and resistance Wide range of bacteriaAnimal cell invasion Salmonella, Shigella, YersiniaCoagulase, hemolysin, enterotoxin StaphylococcusToxins and capsule Bacillus anthracisEnterotoxin, K antigen Escherichia coli




ability) of pathogens that are often plasmid encoded: (1) the abilityof the pathogen to attach to and colonize specific host tissue and(2) the production of toxins, enzymes, and other molecules thatcause damage to the host.

Enteropathogenic strains of Escherichia coli are characterizedby the ability to colonize the small intestine and to produce atoxin that causes diarrhea. Colonization requires a cell surfaceprotein called colonization factor antigen, encoded by a plasmid.This protein confers on bacterial cells the ability to attach toepithelial cells of the intestine. At least two toxins in enteropath-ogenic E. coli are encoded by plasmids: the hemolysin, whichlyses red blood cells, and the enterotoxin, which induces exten-sive secretion of water and salts into the bowel. It is the entero-toxin that is responsible for diarrhea ( Section 27.11).

Some virulence factors are encoded on plasmids. Other viru-lence factors are encoded by other mobile genetic elements,such as transposons and bacteriophages. Some virulence factorsare chromosomal. Several examples are known in which multi-ple virulence genes are present on different genetic elementswithin the same cell. For instance, the genes encoding the viru-lence determinants of Shiga toxin–producing strains of E. coli( Section 36.9) are distributed among the chromosome, abacteriophage, and a plasmid.

BacteriocinsMany bacteria produce proteins that inhibit or kill closely relatedspecies or even different strains of the same species. Theseagents are called bacteriocins to distinguish them from antibi-otics. Bacteriocins have a narrower spectrum of activity than

antibiotics. The genes encoding bacteriocins and the proteinsneeded for processing and transporting them and for conferringimmunity on the producing organism are usually carried on plas-mids or transposons. Bacteriocins are often named after thespecies of organism that produces them. Thus, E. coli producescolicins; Yersinia pestis produces pesticins, and so on.

The Col plasmids of E. coli encode various colicins. Col plas-mids can be either conjugative or nonconjugative. Colicinsreleased from the producer cell bind to specific receptors on thesurface of susceptible cells. The receptors for colicins are typi-cally proteins whose normal function is to transport growth fac-tors or micronutrients across the outer membrane of the cell.Colicins kill cells by disrupting some critical cell function. Manycolicins form channels in the cell membrane that allow potas-sium ions and protons to leak out, leading to loss of the ability togenerate energy. Another major group of colicins are nucleasesand degrade DNA or RNA. For example, colicin E2 is a DNAendonuclease that cleaves DNA, and colicin E3 is a ribonucleasethat cuts at a specific site in 16S rRNA and therefore inactivatesribosomes.

The bacteriocins or bacteriocin-like agents of gram-positive bac-teria are quite different from the colicins but are also often encodedby plasmids; some even have commercial value. For instance, lacticacid bacteria produce the bacteriocin nisin A, which stronglyinhibits the growth of a wide range of gram-positive bacteria and isused as a preservative in the food industry.

MiniQuiz• What properties does an R plasmid confer on its host cell?• What properties does a virulence plasmid typically confer on

its host cell?• How do bacteriocins differ from antibiotics?

III DNA Replication

DNA replication is necessary for cells to divide, whether toreproduce new organisms, as in unicellular microorganisms,

or to produce new cells as part of a multicellular organism. DNAreplication must be sufficiently accurate that the daughter cellsare genetically identical to the mother cell (or almost so). Thisinvolves a host of special enzymes and processes.

6.8 Templates and EnzymesDNA exists in cells as a double helix with complementary basepairing. If the double helix is opened up, a new strand can be syn-thesized as the complement of each parental strand. As shown inFigure 6.13, replication is semiconservative, meaning that thetwo resulting double helices consist of one new strand and oneparental strand. The DNA strand that is used to make a comple-mentary daughter strand is called the template, and in DNAreplication each parental strand is a template for one newly syn-thesized strand (Figure 6.13).

The precursor of each new nucleotide in the DNA strand is adeoxynucleoside 59-triphosphate. The two terminal phosphatesare removed and the innermost phosphate is then attached

mer

sulIS1

IS10

IS1

IS10

tra

tet

94.3/0 kbp

25 kbp

50 kbp

75 kbp

oriT

cat

str

Replicationfunctions

Tn10

Figure 6.12 Genetic map of the resistance plasmid R100. The innercircle shows the size in kilobase pairs. The outer circle shows the locationof major antibiotic resistance genes and other key functions: mer, mer-curic ion resistance; sul, sulfonamide resistance; str, streptomycin resis-tance; cat, chloramphenicol resistance; tet, tetracycline resistance; oriT,origin of conjugative transfer; tra, transfer functions. The locations ofinsertion sequences (IS) and the transposon Tn10 are also shown. Genesfor plasmid replication are found in the region from 88 to 92 kbp.Yrd.Doç.Dr.Yosun MATER (Brock Biology of Microorganisms 13th Edition 2012)

PROKARYOTLARDA DNA DÜZENLENMESİ

• Bakterilerde DNA hücre kuru ağırlığının yaklaşık %2-3’nü oluşturmaktadır.

• DNA’nın ikili sarmal yapısı d e o k s i r i b o z f o s f a t iskeletten oluşur. Her bir imine nukleot id adı verilir.

• Bu birimler purin yada pirimidin bazı, pentoz şeker ve fosfat molekülü içerir.


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Cytosine (C)

NH

O

NH2

N

Thymine (T)

N

NH

O

O

H3C

Uracil(U)

N

NH

O

O

Guanine (G)

N

N

N

NH

NH2

O

N

N

65

4 321

Pyrimidine bases Purine bases

Adenine (A)

N

N

NH2

H

5

67

98

1 2

43

P

O

O–

O–O

C C

O Base

OH

H HHH C

OHRibose

Phosphate

C

CH2

H onlyin DNA

H H

H2C BaseO

O

P O–O

H H H

H

H

H2C BaseO

O

O

5! position

P O–O

O

Nitrogen base attached to 1! ! position

Deoxyribose

(c)

(a)

(b)

H

H

DNA DNA only

RNA only

DNA DNA

5!

4!3! 2!

1!

5!

4!

3! 2!

1!

Phosphodiesterbond

3! position

RNA RNA RNA

Figure 6.1 Components of the nucleic acids. (a) The nitrogen basesof DNA and RNA. Note the numbering system of the rings. In attachingitself to the 19 carbon of the sugar phosphate, a pyrimidine base bondsthrough N-1 and a purine base bonds at N-9. (b) Nucleotide structure.The numbers on the sugar contain a prime (9) after them because therings of the nitrogen bases are also numbered. In DNA a hydrogen ispresent on the 29-carbon of the pentose sugar. In RNA, an OH groupoccupies this position. (c) Part of a DNA chain. The nucleotides are linkedby a phosphodiester bond. In addition to the bases shown, transferRNAs (tRNAs) contain unusual pyrimidines such as pseudouracil anddihydrouracil, and various modified purines not present in other RNAs(see Figure 6.33).

Cells may be regarded as chemical machines and coding devices.As chemical machines, cells transform their vast array of macro-molecules into new cells. As coding devices, they store, process,and use genetic information. Genes and gene expression are thesubject of molecular biology. In particular, the review of molecu-lar biology in this chapter covers the chemical nature of genes,the structure and function of DNA and RNA, and the replicationof DNA. We then consider the synthesis of proteins, macromole-cules that play important roles in both the structure and thefunctioning of the cell. Our focus here is on these processes asthey occur in Bacteria. In particular, Escherichia coli, a member ofthe Bacteria, is the model organism for molecular biology and isthe main example used. Although E. coli was not the first bac-terium to have its chromosome sequenced, this organismremains the best characterized of any organism, prokaryote oreukaryote.

I DNA Structure and GeneticInformation

6.1 Macromolecules and GenesThe functional unit of genetic information is the gene. All lifeforms, including microorganisms, contain genes. Physically,genes are located on chromosomes or other large moleculesknown collectively as genetic elements. Nowadays, in the“genomics era,” biology tends to characterize cells in terms oftheir complement of genes. Thus, if we wish to understand howmicroorganisms function we must understand how genes encodeinformation.

Chemically, genetic information is carried by the nucleic acidsdeoxyribonucleic acid, DNA, and ribonucleic acid, RNA. DNAcarries the genetic blueprint for the cell and RNA is the interme-diary molecule that converts this blueprint into defined aminoacid sequences in proteins. Genetic information consists of thesequence of monomers in the nucleic acids. Thus, in contrast topolysaccharides and lipids, nucleic acids are informationalmacromolecules. Because the sequence of monomers in pro-teins is determined by the sequence of the nucleic acids thatencode them, proteins are also informational macromolecules.

The monomers of nucleic acids are called nucleotides, conse-quently, DNA and RNA are polynucleotides. A nucleotide hasthree components: a pentose sugar, either ribose (in RNA) ordeoxyribose (in DNA), a nitrogen base, and a molecule of phos-phate, PO4

3-. The general structure of nucleotides of both DNAand RNA is very similar (Figure 6.1). The nitrogen bases areeither purines (adenine and guanine) which contain two fusedheterocyclic rings or pyrimidines (thymine, cytosine, and uracil)which contain a single six-membered heterocyclic ring (Figure6.1a). Guanine, adenine, and cytosine are present in both DNAand RNA. With minor exceptions, thymine is present only inDNA and uracil is present only in RNA.

The nitrogen bases are attached to the pentose sugar by aglycosidic linkage between carbon atom 1 of the sugar and anitrogen atom in the base, either nitrogen 1 (in pyrimidine bases)or 9 (in purine bases). A nitrogen base attached to its sugar, but



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Cytosine (C)

NH

O

NH2

N

Thymine (T)

N

NH

O

O

H3C

Uracil(U)

N

NH

O

O

Guanine (G)

N

N

N

NH

NH2

O

N

N

65

4 321

Pyrimidine bases Purine bases

Adenine (A)

N

N

NH2

H

5

67

98

1 2

43

P

O

O–

O–O

C C

O Base

OH

H HHH C

OHRibose

Phosphate

C

CH2

H onlyin DNA

H H

H2C BaseO

O

P O–O

H H H

H

H

H2C BaseO

O

O

5! position

P O–O

O

Nitrogen base attached to 1! ! position

Deoxyribose

(c)

(a)

(b)

H

H

DNA DNA only

RNA only

DNA DNA

5!

4!3! 2!

1!

5!

4!

3! 2!

1!

Phosphodiesterbond

3! position

RNA RNA RNA

Figure 6.1 Components of the nucleic acids. (a) The nitrogen basesof DNA and RNA. Note the numbering system of the rings. In attachingitself to the 19 carbon of the sugar phosphate, a pyrimidine base bondsthrough N-1 and a purine base bonds at N-9. (b) Nucleotide structure.The numbers on the sugar contain a prime (9) after them because therings of the nitrogen bases are also numbered. In DNA a hydrogen ispresent on the 29-carbon of the pentose sugar. In RNA, an OH groupoccupies this position. (c) Part of a DNA chain. The nucleotides are linkedby a phosphodiester bond. In addition to the bases shown, transferRNAs (tRNAs) contain unusual pyrimidines such as pseudouracil anddihydrouracil, and various modified purines not present in other RNAs(see Figure 6.33).

Cells may be regarded as chemical machines and coding devices.As chemical machines, cells transform their vast array of macro-molecules into new cells. As coding devices, they store, process,and use genetic information. Genes and gene expression are thesubject of molecular biology. In particular, the review of molecu-lar biology in this chapter covers the chemical nature of genes,the structure and function of DNA and RNA, and the replicationof DNA. We then consider the synthesis of proteins, macromole-cules that play important roles in both the structure and thefunctioning of the cell. Our focus here is on these processes asthey occur in Bacteria. In particular, Escherichia coli, a member ofthe Bacteria, is the model organism for molecular biology and isthe main example used. Although E. coli was not the first bac-terium to have its chromosome sequenced, this organismremains the best characterized of any organism, prokaryote oreukaryote.

I DNA Structure and GeneticInformation

6.1 Macromolecules and GenesThe functional unit of genetic information is the gene. All lifeforms, including microorganisms, contain genes. Physically,genes are located on chromosomes or other large moleculesknown collectively as genetic elements. Nowadays, in the“genomics era,” biology tends to characterize cells in terms oftheir complement of genes. Thus, if we wish to understand howmicroorganisms function we must understand how genes encodeinformation.

Chemically, genetic information is carried by the nucleic acidsdeoxyribonucleic acid, DNA, and ribonucleic acid, RNA. DNAcarries the genetic blueprint for the cell and RNA is the interme-diary molecule that converts this blueprint into defined aminoacid sequences in proteins. Genetic information consists of thesequence of monomers in the nucleic acids. Thus, in contrast topolysaccharides and lipids, nucleic acids are informationalmacromolecules. Because the sequence of monomers in pro-teins is determined by the sequence of the nucleic acids thatencode them, proteins are also informational macromolecules.

The monomers of nucleic acids are called nucleotides, conse-quently, DNA and RNA are polynucleotides. A nucleotide hasthree components: a pentose sugar, either ribose (in RNA) ordeoxyribose (in DNA), a nitrogen base, and a molecule of phos-phate, PO4

3-. The general structure of nucleotides of both DNAand RNA is very similar (Figure 6.1). The nitrogen bases areeither purines (adenine and guanine) which contain two fusedheterocyclic rings or pyrimidines (thymine, cytosine, and uracil)which contain a single six-membered heterocyclic ring (Figure6.1a). Guanine, adenine, and cytosine are present in both DNAand RNA. With minor exceptions, thymine is present only inDNA and uracil is present only in RNA.

The nitrogen bases are attached to the pentose sugar by aglycosidic linkage between carbon atom 1 of the sugar and anitrogen atom in the base, either nitrogen 1 (in pyrimidine bases)or 9 (in purine bases). A nitrogen base attached to its sugar, but




lacking phosphate, is called a nucleoside. Nucleotides are nucle-osides plus one or more phosphates (Figure 6.1). Nucleotides playother roles in addition to comprising nucleic acids. Nucleotides,especially adenosine triphosphate (ATP) and guanosine triphos-phate (GTP), carry chemical energy. Other nucleotides or deriv-atives function in redox reactions, as carriers of sugars inpolysaccharide synthesis, or as regulatory molecules.

The Nucleic Acids, DNA and RNAThe nucleic acid backbone is a polymer of alternating sugar andphosphate molecules. The nucleotides are covalently bonded byphosphate between the 39- (3 prime) carbon of one sugar and the59-carbon of the next sugar. [Numbers with prime marks refer topositions on the sugar ring; numbers without primes to positionson the rings of the bases.] The phosphate linkage is called aphosphodiester bond because the phosphate connects twosugar molecules by an ester linkage (Figure 6.1). The sequence ofnucleotides in a DNA or RNA molecule is its primary structureand the sequence of bases forms the genetic information.

In the genome of cells, DNA is double-stranded. Each chro-mosome consists of two strands of DNA, with each strand con-taining hundreds of thousands to several million nucleotideslinked by phosphodiester bonds. The strands are held togetherby hydrogen bonds that form between the bases in one strandand those of the other strand. When located next to oneanother, purine and pyrimidine bases can form hydrogen bonds(Figure 6.2). Hydrogen bonding is most stable when guanine (G)bonds with cytosine (C) and adenine (A) bonds with thymine(T). Specific base pairing, A with T and G with C, ensures thatthe two strands of DNA are complementary in base sequence;that is, wherever a G is found in one strand, a C is found in theother, and wherever a T is present in one strand, its complemen-tary strand has an A.

With a few exceptions, all RNA molecules are single-stranded.However, RNA molecules typically fold back upon themselves inregions where complementary base pairing is possible. The termsecondary structure refers to this folding whereas primarystructure refers to the nucleotide sequence. In certain large RNAmolecules, such as ribosomal RNA (Section 6.19), some parts ofthe molecule are unfolded but other regions possess secondarystructure. This leads to highly folded and twisted moleculeswhose biological function depends critically on their final three-dimensional shape.

Genes and the Steps in Information FlowWhen genes are expressed, the information stored in DNA istransferred to ribonucleic acid (RNA). Several classes of RNAexist in cells. Three types of RNA take part in protein synthesis.Messenger RNA (mRNA) is a single-stranded molecule thatcarries the genetic information from DNA to the ribosome, theprotein-synthesizing machine. Transfer RNAs (tRNAs) convertthe genetic information on mRNA into the language of proteins.Ribosomal RNAs (rRNAs) are important catalytic and structuralcomponents of the ribosome. In addition to these, cells contain avariety of small RNAs that regulate the production or activity ofproteins or other RNAs. The molecular processes of geneticinformation flow can be divided into three stages (Figure 6.3):1. Replication. During replication, the DNA double helix is dupli-

cated, producing two double helices.2. Transcription. Transfer of information from DNA to RNA is

called transcription.3. Translation. Synthesis of a protein, using the information

carried by mRNA, is known as translation.

N

N

NN

H NO

NN

O

CH3

H

H

Adenine

N

N

N

N

H O

NN

O

H

HN

NH

H

Cytosine Guanine

Hydrogenbond

Hydrogenbond

Backbone Backbone

BackboneBackbone

Thymine

Figure 6.2 Specific pairing between guanine (G) and cytosine (C)and between adenine (A) and thymine (T) via hydrogen bonds. Theseare the typical base pairs found in double-stranded DNA. Atoms that arefound in the major groove of the double helix and that interact with pro-teins are highlighted in pink. The deoxyribose phosphate backbones of thetwo strands of DNA are also indicated. Note the different shades of greenfor the two strands of DNA, a convention used throughout this book.

PheH2N- Val Asn Gln His Leu

REPLICATION

TRANSCRIPTION OFBOTTOM STRAND

TRANSLATION

Protein

T5! ! T T G T T A A T C A G C T TC A T

A A A C A A T T A G T C G A AG T A

3!

T5!3!

T T G T T A A T C A G C T TC A T


3!5!

T5!3!



3!

5! 3!

5!

3! 5!

-COOH

DNA

RNA U U U G U U A A U C A G C A U C U U

Figure 6.3 Synthesis of the three types of informational macromol-ecules. Note that for any particular gene only one of the two strands ofthe DNA double helix is transcribed.



There is a linear correspondence between the base sequence ofa gene and the amino acid sequence of a polypeptide. Each groupof three bases on an mRNA molecule encodes a single aminoacid, and each such triplet of bases is called a codon. This geneticcode is translated into protein by the ribosomes (which consist ofproteins and rRNA), tRNA, and proteins known as translationfactors.

The three steps shown in Figure 6.3 are used in all cells andconstitute the central dogma of molecular biology (DNA S RNAS protein). Note that many different RNA molecules are eachtranscribed from a relatively short region of the long DNA mole-cule. In eukaryotes, each gene is transcribed to give a singlemRNA (Chapter 7), whereas in prokaryotes a single mRNA maycarry genetic information for several genes, that is, for severalprotein coding regions. Some viruses violate the central dogma(Chapter 9). Some viruses use RNA as the genetic material andmust therefore replicate their RNA using RNA as template. Inretroviruses such as HIV—the causative agent of AIDS—an RNAgenome is converted to a DNA version by a process calledreverse transcription.

MiniQuiz• What components are found in a nucleotide?• How does a nucleoside differ from a nucleotide?• Distinguish between the primary and secondary structure

of RNA.• What three informational macromolecules are involved in genetic

information flow?• In all cells there are three processes involved in genetic

information flow. What are they?

6.2 The Double HelixIn all cells and many viruses, DNA exists as a double-strandedmolecule with two polynucleotide strands whose base sequencesare complementary. (As discussed in Chapter 9, the genomes ofsome DNA viruses are single-stranded.) The complementarity ofDNA arises because of specific base pairing: adenine always pairswith thymine, and guanine always pairs with cytosine. The twostrands of the double-stranded DNA molecule are arranged in anantiparallel fashion (Figure 6.4, distinguished as two shades ofgreen). Thus, the strand on the left runs 59 to 39 from top to bot-toms, whereas the other strand runs 59 to 39 from bottom to top.

The two strands of DNA are wrapped around each other toform a double helix (Figure 6.5) that forms two distinctgrooves, the major groove and the minor groove. Most proteinsthat interact specifically with DNA bind in the major groove,where there is plenty of space. Because the double helix is a reg-ular structure, some atoms of each base are always exposed inthe major groove (and some in the minor groove). Key regionsof nucleotides that are important in interactions with proteinsare shown in Figure 6.2.

Several double-helical structures are possible for DNA. TheWatson and Crick double helix is known as the B-form or B-DNAto distinguish it from the A- and Z-forms. The A-form is shorterand fatter than the B-form. It has 11 base pairs per turn, and the

major groove is narrower and deeper. Double-stranded RNA orhybrids of one RNA plus one DNA strand often form the A-helix.The Z-DNA double helix has 12 base pairs per turn and is left-handed. Its sugar–phosphate backbone is a zigzag line ratherthan a smooth curve. Z-DNA is found in GC- or GT-rich regions,especially when negatively supercoiled. Occasional enzymes andregulatory proteins bind Z-DNA preferentially.

Size and Shape of DNA MoleculesThe size of a DNA molecule is expressed as the number ofnucleotide bases or base pairs per molecule. Thus, a DNA mole-cule with 1000 bases is 1 kilobase (kb) of DNA. If the DNA is adouble helix, then kilobase pairs (kbp) is used. Thus, a doublehelix 5000 base pairs in size would be 5 kbp. The bacteriumEscherichia coli has about 4640 kbp of DNA in its chromosome.When dealing with large genomes the term megabase pair(Mbp) for a million base pairs is used. The genome of E. coli isthus 4.64 Mbp.

Each base pair takes up 0.34 nanometer (nm) in length alongthe double helix, and each turn of the helix contains approximately10 base pairs. Therefore, 1 kbp of DNA is 0.34 !m long with 100helical turns. The E. coli genome is thus 4640 * 0.34 = 1.58 mm

UN

IT 3

H H H

H

H

H2CO

O3!

5!O

P O

H H H

H

H

H2CO

O

O

O–

P O

P O

H H H

H

H

H2CO

O

O

P O

H H H

H

H

H2CO

OH

O

HH

H

H

H2CO

O

O

–O

PO

O

PO

HH

H

H

H2CO

O

PO

HH

H

H

H2CO

O

O

PO

HH

H

H

H2CO

OH

O

–O

–O

–O

–O H

H

H

H

Hydrogen bonds

5!-Phosphate

3!-Hydroxyl

–O

–O

–O

–O

3!-Hydroxyl

5!-Phosphate

Phosphodiesterbond

AT

G

C

TA

C

G

1!

3!

5!

3!

5!

Figure 6.4 DNA structure. Complementary and antiparallel nature of DNA. Note that one chain ends in a 59-phosphate group, whereas the other ends in a 39-hydroxyl. The red bases represent the pyrimidinescytosine (C) and thymine (T), and the yellow bases represent the purinesadenine (A) and guanine (G).Yrd.Doç.Dr.Yosun MATER


• Bazlar ikili sarmal yapıyı oluşturmak için birbirlerine hidrojen bağları (H) ile bağlanırlar. Adenin (A), timin (T) bazına 2H ve guanin (G) ise sitozin (C) bazına 3H bağı ile bağlanır.

• Primidin ve purin bazları hidrofobik bir karaktere ve planer bir yapıya sahiptirler.

• Bazı mikroorganizmalarda (fajlarda) nukleik asitlerin yapısında belirtilen bu bazların yanı sıra minor bazlar olarak adlandırılan, 5’-metil sitozin ve 5’-hidroksi metil urasil bulunmaktadır.

• Yan yana bulunan nukleotid’ler birbirleri ile fosfodiester bağları ile bağlanarak polinukleotid zincirleri, polimerleri oluştururlar.

• Bu bağlantı fosfat grubu pentoz şekerin 5’ pozisyonundaki karbon atomu ile komşu şekerin 3’pozisyonundaki karbon atomu arasında fosfodiester bağı kurmasıyla oluşur. Bağlantı 5’-P ucundan 3’-OH ucuna bağlandığından 5’→3’ fosfodiester bağı adını alır.


• Dört temel bazın (A, T, G, C) değişik sırada yan yana karşılıklı birleşmesinden oluşan DNA iplikleri hücreler ve canlılar için çok önemli olan genetik bilgileri (genomu) taşır. • Bu bilgi baz dizilişi ile ilgilidir. Aynı türün DNA kompozisyonu (diziliş sırası ve sayısı) birbirinin aynıdır, sabittir ve değişmez.

• Genetik düzeyde oluşan değişmeler (mutasyonlar) yeni nesillerde farklı fenotipik karakterde bireylerin meydana gelmesine neden olur. Bu bireylere Mutant adı verilir.

• DNA’nın sarmal ve özellikle çift iplik olması çok önemlidir. Böylece genetik bilgileri güvence altına alır ve bir çoğalma (replikasyon) mekanizması kurarak bir ipliği diğerinin sentezi için kalıp olarak kullanır.

• Transkripsiyonda da sadece bir iplik kullanılır. Diğer iplik replikasyonun doğru yönde ilerlemesine yardımcı olur.


• Polinukleotid ipliklerde bulunan 4 bazın (A, T, G, C) 3 tanesi bir amino asidi (aa) tanımlar.

• Buna üçlü kod sistemi yada Triplet (= Kodon) adı verilir.

• Böylece 20 aa üçlü sistemle 64 farklı kodon oluşturur.

• Böylece her aa en az bir kodonu olması dışında bazı aa birden fazla kod ile tanımlanır.

• Buna ek olarak UAA, UAG ve UGA hiçbir aa tanımlamaz, b u n l a r t r a n s l a s y o n d a sentezlenen peptidin bitişinin s i n y a l i n i v e r e n S T O P kodonlardır.


lacking phosphate, is called a nucleoside. Nucleotides are nucle-osides plus one or more phosphates (Figure 6.1). Nucleotides playother roles in addition to comprising nucleic acids. Nucleotides,especially adenosine triphosphate (ATP) and guanosine triphos-phate (GTP), carry chemical energy. Other nucleotides or deriv-atives function in redox reactions, as carriers of sugars inpolysaccharide synthesis, or as regulatory molecules.

The Nucleic Acids, DNA and RNAThe nucleic acid backbone is a polymer of alternating sugar andphosphate molecules. The nucleotides are covalently bonded byphosphate between the 39- (3 prime) carbon of one sugar and the59-carbon of the next sugar. [Numbers with prime marks refer topositions on the sugar ring; numbers without primes to positionson the rings of the bases.] The phosphate linkage is called aphosphodiester bond because the phosphate connects twosugar molecules by an ester linkage (Figure 6.1). The sequence ofnucleotides in a DNA or RNA molecule is its primary structureand the sequence of bases forms the genetic information.

In the genome of cells, DNA is double-stranded. Each chro-mosome consists of two strands of DNA, with each strand con-taining hundreds of thousands to several million nucleotideslinked by phosphodiester bonds. The strands are held togetherby hydrogen bonds that form between the bases in one strandand those of the other strand. When located next to oneanother, purine and pyrimidine bases can form hydrogen bonds(Figure 6.2). Hydrogen bonding is most stable when guanine (G)bonds with cytosine (C) and adenine (A) bonds with thymine(T). Specific base pairing, A with T and G with C, ensures thatthe two strands of DNA are complementary in base sequence;that is, wherever a G is found in one strand, a C is found in theother, and wherever a T is present in one strand, its complemen-tary strand has an A.

With a few exceptions, all RNA molecules are single-stranded.However, RNA molecules typically fold back upon themselves inregions where complementary base pairing is possible. The termsecondary structure refers to this folding whereas primarystructure refers to the nucleotide sequence. In certain large RNAmolecules, such as ribosomal RNA (Section 6.19), some parts ofthe molecule are unfolded but other regions possess secondarystructure. This leads to highly folded and twisted moleculeswhose biological function depends critically on their final three-dimensional shape.

Genes and the Steps in Information FlowWhen genes are expressed, the information stored in DNA istransferred to ribonucleic acid (RNA). Several classes of RNAexist in cells. Three types of RNA take part in protein synthesis.Messenger RNA (mRNA) is a single-stranded molecule thatcarries the genetic information from DNA to the ribosome, theprotein-synthesizing machine. Transfer RNAs (tRNAs) convertthe genetic information on mRNA into the language of proteins.Ribosomal RNAs (rRNAs) are important catalytic and structuralcomponents of the ribosome. In addition to these, cells contain avariety of small RNAs that regulate the production or activity ofproteins or other RNAs. The molecular processes of geneticinformation flow can be divided into three stages (Figure 6.3):1. Replication. During replication, the DNA double helix is dupli-

cated, producing two double helices.2. Transcription. Transfer of information from DNA to RNA is

called transcription.3. Translation. Synthesis of a protein, using the information

carried by mRNA, is known as translation.

N

N

NN

H NO

NN

O

CH3

H

H

Adenine

N

N

N

N

H O

NN

O

H

HN

NH

H

Cytosine Guanine

Hydrogenbond

Hydrogenbond

Backbone Backbone

BackboneBackbone

Thymine

Figure 6.2 Specific pairing between guanine (G) and cytosine (C)and between adenine (A) and thymine (T) via hydrogen bonds. Theseare the typical base pairs found in double-stranded DNA. Atoms that arefound in the major groove of the double helix and that interact with pro-teins are highlighted in pink. The deoxyribose phosphate backbones of thetwo strands of DNA are also indicated. Note the different shades of greenfor the two strands of DNA, a convention used throughout this book.

PheH2N- Val Asn Gln His Leu

REPLICATION

TRANSCRIPTION OFBOTTOM STRAND

TRANSLATION

Protein

T5! ! T T G T T A A T C A G C T TC A T


3!

T5!3!



3!5!

T5!3!



3!

5! 3!

5!

3! 5!

-COOH

DNA

RNA U U U G U U A A U C A G C A U C U U

Figure 6.3 Synthesis of the three types of informational macromol-ecules. Note that for any particular gene only one of the two strands ofthe DNA double helix is transcribed.



Table 6.5 The genetic code as expressed by triplet base sequences of mRNA

Codon Amino acid Codon Amino acid Codon Amino acid Codon Amino acid

UUU Phenylalanine UCU Serine UAU Tyrosine UGU CysteineUUC Phenylalanine UCC Serine UAC Tyrosine UGC CysteineUUA Leucine UCA Serine UAA None (stop signal) UGA None (stop signal)UUG Leucine UCG Serine UAG None (stop signal) UGG Tryptophan

CUU Leucine CCU Proline CAU Histidine CGU ArginineCUC Leucine CCC Proline CAC Histidine CGC ArginineCUA Leucine CCA Proline CAA Glutamine CGA ArginineCUG Leucine CCG Proline CAG Glutamine CGG Arginine

AUU Isoleucine ACU Threonine AAU Asparagine AGU SerineAUC Isoleucine ACC Threonine AAC Asparagine AGC SerineAUA Isoleucine ACA Threonine AAA Lysine AGA ArginineAUG (start)a Methionine ACG Threonine AAG Lysine AGG Arginine

GUU Valine GCU Alanine GAU Aspartic acid GGU GlycineGUC Valine GCC Alanine GAC Aspartic acid GGC GlycineGUA Valine GCA Alanine GAA Glutamic acid GGA GlycineGUG Valine GCG Alanine GAG Glutamic acid GGG Glycine

aAUG encodes N–formylmethionine at the beginning of polypeptide chains of Bacteria.

as the genetic code. A triplet of three bases called a codonencodes each specific amino acid. The 64 possible codons (fourbases taken three at a time = 43) of mRNA are shown in Table6.5. The genetic code is written as RNA rather than as DNAbecause it is mRNA that is translated. Note that in addition to thecodons for amino acids, there are also specific codons for startingand stopping translation.

Properties of the Genetic CodeThere are 22 amino acids that are encoded by the genetic infor-mation carried on mRNA. (A variety of others are created bymodification of these after translation.) Consequently, becausethere are 64 codons, many amino acids are encoded by more thanone codon. Although knowing the codon at a given locationunambiguously identifies the corresponding amino acid, thereverse is not true. Knowing the amino acid does not mean thatthe codon at that location is known. A code such as this that lacksone-to-one correspondence between “word” (that is, the aminoacid) and code is called a degenerate code. However, knowing theDNA sequence and the correct reading frame, one can specifythe amino acid sequence of a protein. This permits the determi-nation of amino acid sequences from DNA base sequences and isat the heart of genomics (Chapter 12). In most cases where multi-ple codons encode the same amino acid, the multiple codons areclosely related in base sequence (Table 6.5).

A codon is recognized by specific base pairing with a comple-mentary sequence of three bases called the anticodon, which isfound on tRNAs. If this base pairing were always the standardpairing of A with U and G with C, then at least one specific tRNAwould be needed to recognize each codon. In some cases, this istrue. For instance, there are six different tRNAs in Escherichiacoli for the amino acid leucine, one for each codon (Table 6.5). Bycontrast, some tRNAs can recognize more than one codon. Thus,although there are two lysine codons in E. coli, there is only onelysyl tRNA whose anticodon can base-pair with either AAA or

AAG. In these special cases, tRNA molecules form standard basepairs at only the first two positions of the codon while toleratingirregular base pairing at the third position. This phenomenon iscalled wobble and is illustrated in Figure 6.31, where a pairingbetween G and U (rather than G with C) is illustrated at the wob-ble position.

Stop and Start CodonsA few codons do not encode any amino acid (Table 6.5). Thesecodons (UAA, UAG, and UGA) are the stop codons, and theysignal the termination of translation of a protein-codingsequence on the mRNA. Stop codons are also called nonsensecodons, because they interrupt the “sense” of the growingpolypeptide when they terminate translation.

A coding sequence on messenger RNA is translated beginningwith the start codon (AUG), which encodes a chemically modi-fied methionine, N-formylmethionine. Although AUG at thebeginning of a coding region encodes N-formylmethionine, AUG

Key bases incodon:anticodon pairing

Wobble position; base pairing moreflexible here

AlaninetRNA

Anticodon

Codon

mRNA

C G G

G C U

5!

5! 3!

3!

Figure 6.31 The wobble concept. Base pairing is more flexible for thethird base of the codon than for the first two. Only a portion of the tRNA isshown here.



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(a)—General structure of an amino acid

(b)—Structure of the amino acid “R” groups

(Note: Because proline lacks a free amino group,the entire structure of this amino acid is shown, not just the R group).

H2

CH2HO Ser Serine (S)

CHCH3

OH

Thr Threonine (T)

CH2C

O

OH2N Asn Asparagine (N)

CH2C CH2H2N Gln Glutamine (Q)

CH2HS Cys Cysteine (C)

CH2HO Tyr Tyrosine (Y)

CH2HSe Sec Selenocysteine(U)

H Gly Glycine (G)

N

CH2H2C

H2C CH COO–Pro Proline (P)

H

CHCH3

CH3

Val Valine (V)

CH2CHCH3

CH3

Leu Leucine (L)

CHCH3

CH2CH3

Ile Isoleucine (I)

CH2 Phe Phenylalanine (F)

CH2

NH

Trp Tryptophan(W)

CH3 Ala Alanine (A)

CH2C

O

C

O

-O CH2 Glu Glutamate (E)

CH2

O-O Asp Aspartate (D)C

(CH2)4

CH2 CH2CH2CH2+NH3 Lys Lysine (K)

CH2CH2CH2

H3CH2C

C

+NH2 NH

N

N

HH

C

CC

NH2

Arg Arginine (R)

Pyl Pyrrolysine (O)

CH2

HN

+HN His Histidine (H)

Met Methionine(M)CH2S CH2CH3

OHC

O

C

R

H

H2N

Amino group Carboxylic acid group

!-Carbon

Ionizable: acidic

Ionizable: basic

Nonionizable polar

Nonpolar(hydrophobic)

Figure 6.29 Structure of the 22 genetically encoded amino acids. (a) General structure. (b) R groupstructure. The three-letter codes for the amino acids are to the left of the names, and the one-letter codesare in parentheses to the right of the names. Pyrrolysine has thus far been found only in certainmethanogenic Archaea ( Section 19.3).

H2N C C

H O

OH NH

C C

H

R2

O

OH+ H

H2O

H2N C C

H

R1

O

N C C

H

R2

O

OH

Peptide bond

R1

HN-terminus C-terminus

Figure 6.30 Peptide bond formation. R1 and R2 refer to the sidechains of the amino acids. Note that, following peptide bond formation, a free OH group is present at the C-terminus for formation of the nextpeptide bond.

acids contain hydrophobic side chains and are known as nonpo-lar amino acids. Cysteine contains a sulfhydryl group (–SH).Using their sulfhydryl groups, two cysteines can form a disulfidelinkage (R–S–S–R) that connects two polypeptide chains.

The diversity of chemically distinct amino acids makes possi-ble an enormous number of unique proteins with widely differentbiochemical properties. If one assumes that an average polypep-tide contains 300 amino acids, there are 22300 different polypep-

tide sequences that are theoretically possible. No cell has any-where near this many different proteins. In practice, a cell ofEscherichia coli contains around 2000 different kinds of proteins.

The linear sequence of amino acids in a polypeptide is theprimary structure. This, ultimately, determines the further fold-ing of the polypeptide, which in turn determines the biologicalactivity. The two ends of a polypeptide are designated as the“C-terminus” and “N-terminus” depending on whether a free car-boxylic acid group or a free amino group is found (Figure 6.30).

MiniQuiz• What chemical groups do amino acids contain?• Draw the structure of a dipeptide containing the amino acids

alanine and tyrosine. Outline the peptide bond.• Which enantiomeric form of amino acids is found in proteins?• Glycine does not have two different enantiomers; why?

6.17 Translation and the Genetic CodeIn the first two steps in biological information transfer, replica-tion and transcription, nucleic acids are synthesized on nucleicacid templates. The last step, translation, also uses a nucleic acidas template, but in this case the product is a protein rather than anucleic acid. The heart of biological information transfer is thecorrespondence between the nucleic acid template and theamino acid sequence of the polypeptide product. This is known


Bakterilerde genetik kodlar ve özelliklerini özetlersek; 1.Kodlar triplet’dirler; genetik bilgiyi saklayan kodlar, 3 bazın birleşmesinden meydana gelmişlerdir. 2.Kodlar değişkendir; her aa için birden fazla triplet bulunmaktadır. 3.Kodlar birbiri ile çakışmazlar; yani bir triplet sadece bir aa tanımlar. Yan yana bulunan bazlardan biri, bir önceki veya bir sonraki aa bir parçası değildir. Dolayısıyla kodonlar birbirinden bağımsızdır. 4.Kodlar arasında boşluk yoktur. Yani bakterilerde kodonlar DNA veya mRNA üzerinde yan yana aralıksız olarak devam ederler. Diğer bir değişle bakterilerde viruslarda ve ökaryotlarda olan iki gen arasında 2000 yada daha fazla sayıda okunmayan baz sıraları (=intron) yoktur. 5.Kodlar evrenseldir; Bakterilerde bir kodon hangi aa tanımlıyorsa ökaryotlarda da aynı amino asidi tanımlar.


Bakterilerde DNA Formasyonları Bakterilerde DNA başlıca üç yapısal (Topolojik) formda bulunur. 1.Doğrusal (=Lineer) DNA formu bazı fajlarda (lambda, T2, T4, T6, T1, T3 vs) ve viruslerde (adenovirididae vs) genetik materyali oluşturan sarmal DNA iki uçu açık doğrusal formda bulunur. 2.Dairesel DNA formu; bakteriler, bazı fajlar (PM2 vs), viruslar (popavaviridae vs) ve plazmitlerde bulunur. 3.Süper sarmal (süperheliks) DNA formu; fajlarda, plazmit ve virüslerde daha fazla rastlanan bu form çift sarmal DNA’nın serbest ortamda kendi etrafında 360° dönmesiyle oluşur. • Süper sarmalın sola dönüşlü olanı pozitif süper sarmal veya sağa dönüşlü negatif süper sarmal formunda bulunabilir.


• İn vivo koşullarda genellikle negatif süpersarmala rastlanır.

• Pozitif süpersarmal ise daha sağlam ve sıkı yapısal bir özellik taşımaktadır.

• Süpersarmal bakteri DNA’sının santrifüj edildiğinde daha çabuk çökmesini sağlar.

• Süpersarmalların varlığı birkaç yöntemle ortaya koyulabilir;

1.Elektron mikroskobunda doğrudan kontrolle fotoraflanarak belirlenmiş pek çok örnek vardır. 2.Sedimentasyon yöntemi ile süpersarmal DNA’ların daha hızlı çöktüğü belirlenmiştir. 3.Elektroforez yöntemi ile doğrusal DNA iplikleri süpersarmal DNA’lardan daha başlangıç noktasına yakın yer alır.


• DNA’nın süpersarmal durumuna gelmesi ve açılmasında Topoizomeraz enzimleri görevlidir. Bilinen iki tipi vardır.

Topoizomeraz I: 100.000 molekül ağırlığında ve monomerik yapıda olan bu enzim negatif süpersarmal DNA’nın tekbir ipliğinin açılmasında görev alır. Bu fonksiyonu için enerjiye gereksinim duymaz.

Topoizomeraz II: Bu enzim ATP’den enerji alarak süpersarmal DNA’nın her iki iplikçiğinde açılmalara ve kopmalara yol açar. •  E. coli’de yer alan ve topoizomeraz II ailesinden sayılan DNA giraz dinlenme halindeki DNA’da negatif süpersarmallar oluşmasına yol açar.

• DNA Giraz: 400.000 molekül ağırlığında tetramerik yapıdadır (A2B2). Hücrelerde A alt biriminin B’den 10 defa fazla bulunduğu gösterilmiştir.

• 30°C’ta her dakikada 100 süper sarmal oluşturabilir. Bu enzim aynı zamanda bakterilerde replikasyonun ilerlemesinde de önemli rollere sahiptir. Bazı antibiyotiklerin bu enzimi inhibe ettiği bilinmektedir. Bunlar;


Giraz A nalidixic asit, oxilinic asit ve Giraz B coumermycin ve novobiocin tarafından inhibe edilir. • Bakterilerdeki DNA’nın uzunluğu bakıldığında ise son derece değişken olduğu bulunmuştur.

• E.coli’de DNA’nın uzunluğu 1,1-1,5 mm kadardır. Bu bakterinin boyunun 400-500 katı kadardır. • 4106 nükleotid çiftinden oluşur ve molekül ağırlığı 2109 Daltondur.

• E.coli’de yaklaşık 3000 gen yer alır. 1000 tanesinden fazlasının yeri bilinmektedir.



axis in the opposite sense from the right-handed double helix.Negatively supercoiled DNA is the form predominantly found innature. However, certain species of Archaea (Chapter 7) thatgrow at very high temperatures do contain positively supercoiledDNA. In Escherichia coli more than 100 supercoiled domains arethought to exist, each of which is stabilized by binding to specificproteins.

Topoisomerases: DNA GyraseSupercoils are inserted or removed by enzymes known as topo-isomerases. Two major classes of topoisomerase exist with differ-ent mechanisms. Class I topoisomerases make a single-strandedbreak in the DNA that allows the rotation of one strand of thedouble helix around the other. Each rotation adds or removes asingle supercoil. After this, the nick is resealed. For example, sur-plus supercoiling in DNA is generally removed by the class Ienzyme, topoisomerase I. As shown in Figure 6.8, a break in thebackbone (a nick) of either strand allows DNA to lose its super-coiling. However, to prevent the entire bacterial chromosomefrom becoming relaxed every time a nick is made, the chromo-some contains supercoiled domains as shown in Figure 6.8d. Anick in the DNA of one domain does not relax DNA in the oth-ers. It is unclear precisely how these domains are formed,although specific DNA-binding proteins are involved.

Class II topoisomerases make double-stranded breaks, pass thedouble helix through the break, and reseal the break (Figure 6.9).Each such operation adds or removes two supercoils. Insertingsupercoils into DNA requires energy from ATP, whereas releasingsupercoils does not. In Bacteria and most Archaea, the class IItopoisomerase, DNA gyrase, inserts negative supercoils intoDNA. Some antibiotics inhibit the activity of DNA gyrase. Theseinclude the quinolones (such as nalidixic acid), the fluoro-quinolones (such as ciprofloxacin), and novobiocin.

Through the activity of topoisomerases, a DNA molecule canbe alternately supercoiled and relaxed. Supercoiling is necessaryfor packing the DNA into the cell and relaxation is necessary forDNA replication and transcription. In most prokaryotes, thelevel of negative supercoiling results from the balance between

the activity of DNA gyrase and topoisomerase I. Supercoilingalso affects gene expression. Certain genes are more activelytranscribed when DNA is supercoiled, whereas transcription ofother genes is inhibited by supercoiling.

MiniQuiz• Why is supercoiling important?• What mechanism is used by DNA gyrase?• What function do topoisomerases serve inside cells?

6.4 Chromosomes and Other Genetic Elements

Structures containing genetic material (DNA in most organisms,but RNA in some viruses) are called genetic elements. Thegenome is the total complement of genes in a cell or virus.Although the main genetic element in prokaryotes is thechromosome, other genetic elements are found and play impor-tant roles in gene function in both prokaryotes and eukaryotes(Table 6.1). These include virus genomes, plasmids, organellargenomes, and transposable elements. A typical prokaryote has asingle circular chromosome containing all (or most) of the genesfound inside the cell. Although a single chromosome is the ruleamong prokaryotes, there are exceptions. A few prokaryotes con-tain two chromosomes. Eukaryotes have multiple chromosomesmaking up their genome ( Section 7.5). Also, the DNA in allknown eukaryotic chromosomes is linear in contrast to mostprokaryotic chromosomes, which are circular DNA molecules.

Viruses and PlasmidsViruses contain genomes, either of DNA or RNA, that controltheir own replication and their transfer from cell to cell. Both lin-ear and circular viral genomes are known. In addition, the nucleicacid in viral genomes may be single-stranded or double-stranded.Viruses are of special interest because they often cause disease.We discuss viruses in Chapters 9 and 21 and a variety of viral dis-eases in later chapters.

Relaxed circle

One part of circle is laid over the other

Helix makes contact in two places

DNA gyrase makesdouble-strand break

Double-strandbreak resealed

Unbroken helix is passed through the break

Following DNA gyrase activity, two negative supercoils result

Supercoiled DNA

Figure 6.9 DNA gyrase. Introduction of negative supercoiling into circular DNA by the activity of DNAgyrase (topoisomerase II), which makes double-strand breaks.


PROKARYOTLARDA RNA DÜZENLENMESİ • Mikroorganizmalarda DNA kadar önemli olan bir diğer makromolekül de ribonükleik asittir. • DNA gibi pirimidin veya pürin bazı, pentoz (D-riboz) şeker ve fosfat molekülünden oluşur. • RNA’nın yapısında yer alan pirimidin bazlarından Timin yerine Urasil yer alır. • Yapı olarak birbirine çok benzeyen bu iki azotlu baz arasında tek fark timinde 5’pozisyonunda yer alan metil gurubu (CH3) yerine hidrojen (H) yer almasıdır. • RNA tiplerine baktığımızda, canlılarda 4 farklı şekilde bulunduğunu görürüz. Bunlar; 1.Mesenger (Mesajcı) RNA (mRNA) 2.Transfer RNA (tRNA) 3.Ribozomal RNA (rRNA) 4.Primer RNA (pRNA)


1.Mesenger (Mesajcı) RNA (mRNA): DNA ipliklerinden birinde yer alan RNA polimeraz enziminin katalitik etkisi ile genetik bilgileri mRNA’ya aktarılır (Transkripsiyon). • Yeni oluşan mRNA kendisine kalıplık yapan DNA iplikçiğine antiparalel bir durum gösterir. • Hücrede mRNA’nın ömrü 2-3 dk kadardır, RNaze’lar ile hemen sindirilir. • Yapısına bakıldığında 5’ucunda translokasyonu başlatma sinyali veren 30S’lik ribosomal alt üniteye bağlanan AUG kodonu bulunur. • Bu başlama kodonundan sola doğru Shine Dalgarno (SD) dizisi diye bilinen nükleotid sıraları bulunmaktadır. • Bunlar mRNA’nın 30S ribozom alt birimine daha sıkı bağlanmasın da görev alır. • mRNA’nın iç kısmında yer alan AUG kodonu ise met tRNA’nın özel bağlanma yeridir.


2.Transfer RNA (tRNA): Protein sentezinde amino açil sentetaz ile aktive olduktan sonra mRNA’da ki üçlü kodona uygun amino asitleri ribozomlara taşıyan moleküldür. Her aa uygun bir veya birden fazla tRNA vardır. Morfolojisi 4 yapraklı yoncaya benzer burada en önemli kol antikodon içeren koldur.



MiniQuiz• What are stop codons and start codons?• Why is it important for the ribosome to read “in frame”?• What is codon bias?• If you were given a nucleotide sequence, how would you

find ORFs?

6.18 Transfer RNAA transfer RNA carries the anticodon that base-pairs with thecodon on mRNA. In addition, each tRNA is specific for the aminoacid that corresponds to its own anticodon (that is, the cognateamino acid). The tRNA and its specific amino acid are linked byspecific enzymes called aminoacyl-tRNA synthetases. Theseensure that a particular tRNA receives the correct amino acidand must thus recognize both the tRNA and its cognate aminoacid.

General Structure of tRNAThere are about 60 different tRNAs in bacterial cells and100–110 in mammalian cells. Transfer RNA molecules are short,single-stranded molecules that contain extensive secondarystructure and have lengths of 73–93 nucleotides. Certain basesand secondary structures are constant for all tRNAs, whereasother parts are variable. Transfer RNA molecules also containsome purine and pyrimidine bases that differ somewhat from thestandard bases found in RNA because they are chemically modi-fied. These modifications are made to the bases after transcrip-tion. These unusual bases include pseudouridine (!), inosine,

dihydrouridine (D), ribothymidine, methyl guanosine, dimethylguanosine, and methyl inosine. The mature and active tRNA alsocontains extensive double-stranded regions within the molecule.This secondary structure forms by internal base pairing when thesingle-stranded molecule folds back on itself (Figure 6.33).

The structure of a tRNA can be drawn in a cloverleaf fashion,as in Figure 6.33a. Some regions of tRNA secondary structure arenamed after the modified bases found there (the T!C and Dloops) or after their functions (anticodon loop and acceptorstem). The three-dimensional structure of a tRNA is shown inFigure 6.33b. Note that bases that appear widely separated in thecloverleaf model may actually be much closer together whenviewed in three dimensions. This allows some of the bases in oneloop to pair with bases in another loop.

The Anticodon and the Amino Acid–Binding SiteOne of the key variable parts of the tRNA molecule is the anti-codon, the group of three bases that recognizes the codon on themRNA. The anticodon is found in the anticodon loop (Figure6.33). The three nucleotides of the anticodon recognize thecodon by specifically pairing with its three bases. By contrast,other portions of the tRNA interact with both the rRNA and pro-tein components of the ribsome, nonribosomal translation pro-teins, and the aminoacyl synthetase enzyme.

At the 39 end, or acceptor stem, of all tRNAs are threeunpaired nucleotides. The sequence of these three nucleotides isalways cytosine-cytosine-adenine (CCA), and they are absolutelyessential for function. Curiously, in most organisms these threenucleotides are not encoded by the tRNA genes on the chromo-some. Instead they are added, one after another, by an enzyme

A

Acceptor end

phe

CCACGCUUAA

GCGGAUUU

AmGC U C AG

Acceptor stem

D loop

DDGGG

AGAGC

Anticodon stem

mGCCAGAmC

U

U U

YmGAA

AY

mCUGGAmGU

CmCUGUG

T!CG

mAU

C C A C A G

T!C loopG

C mRNA

Acceptor stem

Acceptor endT!C loop

D loop

Anticodon stem

Anticodon loop mGAnticodon

(b)(a)

A A

Codon

Anticodon5"

5"

3"

3"5"

3"

Figure 6.33 Structure of a transfer RNA. (a) The conventional cloverleaf structural drawing of yeastphenylalanine tRNA. The amino acid is attached to the ribose of the terminal A at the acceptor end. A, ade-nine; C, cytosine; U, uracil; G, guanine; T, thymine; !, pseudouracil; D, dihydrouracil; m, methyl; Y, a modi-fied purine. (b) In fact, the tRNA molecule folds so that the D loop and T!C loops are close together andassociate by hydrophobic interactions.


PO C

C

HNH 2

CHCH 3

CH 3O

O CC

HNH 2

CHCH 3

CH 3O

OH

(a) (b)

Anticodon region

Aminoacyl-tRNAsynthetase for valine

AMP

AMP

Valine

Amino acid(valine)

UnchargedtRNA-specificfor valine (tRNAVal )

Charged valyl tRNA,ready for proteinsynthesis

Linkage of valineto tRNAVal

Din

o M

oras

tRNA acceptor stem

Anticodon loop

5! 3!

CA

C

CA

C

Figure 6.34 Aminoacyl-tRNA synthetase. (a) Mode of activity of an aminoacyl-tRNA synthetase.Recognition of the correct tRNA by a particular synthetase involves contacts between specific nucleic acidsequences in the D loop and acceptor stem of the tRNA and specific amino acids of the synthetase. In thisdiagram, valyl-tRNA synthetase is shown catalyzing the final step of the reaction, where the valine in valyl-AMP is transferred to tRNA. (b) A computer model showing the interaction of glutaminyl-tRNA synthetase(blue) with its tRNA (red). Reprinted with permission from M. Ruff et al. 1991. Science 252: 1682–1689. © 1991, AAAS.


UN

IT 3

called the CCA-adding enzyme, using CTP and ATP as sub-strates. The cognate amino acid is covalently attached to the ter-minal adenosine of the CCA end by an ester linkage to the ribosesugar. As we shall see, from this location on the tRNA, the aminoacid is incorporated into the growing polypeptide chain on theribosome by a mechanism described in the next section.

Recognition, Activation, and Charging of tRNAsRecognition of the correct tRNA by an aminoacyl-tRNA syn-thetase involves specific contacts between key regions of thetRNA and the synthetase (Figure 6.34). As might be expectedbecause of its unique sequence, the anticodon of the tRNA isimportant in recognition by the synthetase. However, other con-tact sites between the tRNA and the synthetase are also impor-tant. Studies of tRNA binding to aminoacyl-tRNA synthetases, inwhich specific tRNA bases have been changed by mutation, haveshown that only a small number of key nucleotides in tRNA areinvolved in recognition. These other key recognition nucleotides

are often part of the acceptor stem or D loop of the tRNA (Figure6.33). It should be emphasized that the fidelity of this recognitionprocess is crucial, for if the wrong amino acid is attached to thetRNA, it will be inserted into the growing polypeptide, likelyleading to the synthesis of a faulty protein.

The specific reaction between amino acid and tRNA catalyzedby the aminoacyl-tRNA synthetase begins with activation of theamino acid by reaction with ATP:

Amino acid + ATP g aminoacyl—AMP + P—PThe aminoacyl-AMP intermediate formed normally remainsbound to the enzyme until collision with the appropriate tRNAmolecule. Then, as shown in Figure 6.34a, the activated aminoacid is attached to the tRNA to form a charged tRNA:

Aminoacyl—AMP + tRNA g aminoacyl—tRNA + AMPThe pyrophosphate (PPi) formed in the first reaction is split by apyrophosphatase, giving two molecules of inorganic phosphate.


3 . R i b o z o m a l R N A ( r R N A ) : ribozomların aa ile birleşmesinde ve proteine dönüşmesinde rol alır. • Ribozomların %60 RNA ve %40 özel proteinlerdir. • E.coli’de başlıca 3 tip rRNA bulunur. Bunlar 5S rRNA, 23S rRNA, 16S rRNA’dır. 4.Pr imer RNA (pRNA): DNA eşlenmesi sırasında iplikciğin 5’→3’ yönünde sentezlenmesini sağlar. Ters iplikçikte yön 3’←5’ ama ters olacağı için burada kesintili sentez yap ı l ı r o luşan bu k ı sa DNA parçalarına Okozaki parçaları adı verilir. • Bu parçaların araları polimeraz I enzimi yardımıyla doldurulup, DNA ligaz ile birleştirilir.


RNA

Primary transcript

Maturetranscript

DNA

3!

Transc

riptio

n

terminato

r

Gene e

ncoding

5S rRNA

Gene e

ncoding

23S rRNA

Gene e

ncoding

16S rRNA

Gene e

ncoding

a tRNA

Promoter

16S rRNA 23S rRNA 5S rRNAtRNA

Spacers

5!

DegradationProcessingto remove spacers

Figure 6.28 A ribosomal rRNA transcription unit from Bacteria andits subsequent processing. In Bacteria all rRNA transcription units havethe genes in the order 16S rRNA, 23S rRNA, and 5S rRNA (shownapproximately to scale). Note that in this particular transcription unit thespacer between the 16S and 23S rRNA genes contains a tRNA gene. In other transcription units this region may contain more than one tRNAgene. Often one or more tRNA genes also follow the 5S rRNA gene andare cotranscribed. Escherichia coli contains seven rRNA transcriptionunits.

These cotranscribed transcripts must be processed by cuttinginto individual units to yield mature (functional) rRNAs ortRNAs. Overall, RNA processing is rare in prokaryotes but com-mon in eukaryotes, as we will see later (Chapter 7).

In prokaryotes, most messenger RNAs have a short half-life(on the order of a few minutes), after which they are degradedby cellular ribonucleases. This is in contrast to rRNA andtRNA, which are stable RNAs. This stability is due to tRNAsand rRNAs forming highly folded structures that prevent themfrom being degraded by ribonucleases. By contrast, normalmRNA does not form such structures and is susceptible toribonuclease attack. The rapid turnover of prokaryotic mRNAspermits the cell to quickly adapt to new environmental condi-tions and halt translation of messages whose products are nolonger needed.

Polycistronic mRNA and the OperonIn prokaryotes, genes encoding related enzymes are often clus-tered together. RNA polymerase proceeds through such clustersand transcribes the whole group of genes into a single, longmRNA molecule. An mRNA encoding such a group of cotran-scribed genes is called a polycistronic mRNA. When this is trans-lated, several polypeptides are synthesized, one after another, bythe same ribosome.

A group of related genes that are transcribed together to give asingle polycistronic mRNA is known as an operon. Assemblinggenes for the same biochemical pathway or genes needed underthe same conditions into an operon allows their expression to becoordinated. Despite this, eukaryotes do not have operons andpolycistronic mRNA (Chapter 7). Often, transcription of an

operon is controlled by a specific region of the DNA found justupstream of the protein-coding region of the operon. This is con-sidered in more detail in Chapter 8.

MiniQuiz• What is the role of messenger RNA (mRNA)?• What is a transcription unit?• What is a polycistronic mRNA?• What are operons and why are they useful to prokaryotes?

V Protein Structure and Synthesis

6.16 Polypeptides, Amino Acids, and the Peptide Bond

Proteins play major roles in cell function. Two major classes ofproteins are catalytic proteins (enzymes) and structural proteins.Enzymes are the catalysts for chemical reactions that occur incells. Structural proteins are integral parts of the major struc-tures of the cell: membranes, walls, ribosomes, and so on. Regu-latory proteins control most cell processes by a variety ofmechanisms, including binding to DNA. However, all proteinsshow certain basic features in common.

Proteins are polymers of amino acids. All amino acids containan amino group (–NH2) and a carboxylic acid group (–COOH)that are attached to the !-carbon (Figure 6.29a). Linkagesbetween the carboxyl carbon of one amino acid and the aminonitrogen of a second (with elimination of water) are known aspeptide bonds (Figure 6.30). Two amino acids bonded by pep-tide linkage constitute a dipeptide; three amino acids, a tripep-tide; and so on. When many amino acids are linked they form apolypeptide. A protein consists of one or more polypeptides.The number of amino acids differs greatly from one protein toanother, from as few as 15 to as many as 10,000.

Each amino acid has a unique side chain (abbreviated R).These vary considerably, from as simple as a hydrogen atom inthe amino acid glycine to aromatic rings in phenylalanine, tyro-sine, and tryptophan (Figure 6.29b). Amino acids exist as pairs ofenantiomers. These are optical isomers that have the samemolecular and structural formulas, except that they are mirrorimages and are designated as either D or L, depending on whethera pure solution rotates light to the right or left, respectively. Nat-ural proteins employ L-amino acids only. Nevertheless, D-aminoacids are occasionally found in cells, most notably in the cell wallpolymer peptidoglycan ( Section 3.6) and in certain peptideantibiotics ( Section 26.9). Cells can interconvert certainenantiomers by enzymes called racemases.

The chemical properties of an amino acid are governed by itsside chain. Amino acids with similar chemical properties aregrouped into related “families” (Figure 6.29b). For example, theside chain may contain a carboxylic acid group, as in aspartic acidor glutamic acid, rendering the amino acid acidic. Others containadditional amino groups, making them basic. Several amino


DNA‘NIN FONKSİYONLARI Hücre içinde DNA’nın 5 tür temel işlevi bulunmaktadır. Bunlar; 1.DNA Replikasyonu 2.Transkripsiyon 3.Revers Transkripsiyon 4.DNA Tamir Mekanizması 5.DNA Rekombinasyonları


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