bio 108 lec 7
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Protein Synthesis,
Processing andRegulation
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Translation
-the synthesis of proteins as directed by mRNA templates.
-carried out on ribosomes
-Protein synthesis is thus the final stage of geneexpression.
involves interactions between three types of RNA
molecules (mRNA templates, tRNAs, and rRNAs), as wellas various proteins that are required for translation
basic mechanics are also the same in all cells.
polypeptide chains are synthesized from the amino to
the carboxy terminusEach amino acid is specified by three bases (a codon) in
the mRNA, according to a nearly universal genetic code.
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1) mRNA transmits information present in DNA
2) tRNA - acts a bilingual translator molecule
3) Ribosomes and rRNA the workbenches for
protein synthesis4) tRNA synthetases - serve to mediate the
attachment of specific amino acids to specific
tRNA molecules.
5)amino acids
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Transfer RNAs serve as adaptors that align amino acids on the
mRNA template. Aminoacyl tRNA synthetases attach amino acids
to the appropriate tRNAs, which then bind to mRNA codons bycomplementary base pairing.
Transfer RNAs:
Anticodon- The nucleotide sequence oftransfer RNA that forms complementary
base pairs with a codon sequence onmessenger RNA.
aminoacyl tRNA synthetase- An enzymethat joins a specific amino acid to a tRNA
molecule carrying the correct anticodon
sequence.
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Figure 7.2. Attachment of
amino acids to tRNAs
Activated form
of amino acid
Anticodon
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Common structural components:
1. The 3 terminal-CCAsequence to which the aminoacid is bound. The encoded aminoacid covalently attaches to theribose of the terminal adenosine.
The other end of the moleculepossesses a 3 nucleotide sequence
called the anticodon which is theregion of the molecule that attaches
to the mRNA by complimentary basepairing.
2. A conserved cloverleaf
secondary structure
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Two-step activation of amino acids
The 3'-end
adenine of
a tRNA
This ester
bond gets
readily
hydrolyzed
unlessprotected
by a factor.
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The Ribosome:
particles composed of RNA and proteins that are the
sites of protein synthesis.
The 4-letter language in mRNA is translated
into 20-letter language in protein by
ribosome.
Ribosomes in
the cytoplasm
Ribosomes onER membrane
Three binding sites ofaa-tRNA across largeand small subunits
A passage for mRNA
A tunnel for emerging
peptide
Figure 7.4.Ribosome structure
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facilitate the coupling of the tRNA anticodons with mRNA codons.
Each ribosome has a large and a small subunit formed in the
nucleolus.
Ribosome is composed of proteins and ribosomal RNA (rRNA), the most
abundant RNA in the cell. rRNA is transcribed in the nucleus, then bind to
special proteins to form the ribosomal subunits in the nucleolus.
The large and small subunits join to form a functional ribosome only
when they attach to an mRNA molecule.
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Eukaryotes Prokaryotes
encodes a single polypeptide
(monocistric)
the 5 end is capped (with 7
methylguanusine
the 5- nontranslated region
separates the cap from the
translational initiation signal
encodes more than one
polypeptide (polycistronic)
The 5 end is not capped
initiation codons are
preceded by the a specific
nucleotide sequence called
the Shine-Delgarnosequence.
Both prokaryotic and eukaryotic mRNAs have 5 and 3 non coding sequences.
These are called the 5 UTR and 3 UTR.
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* Two amino acids are designated by single codons: methionine by AUG andtryptophan by UGG
The rest are designated by two, three, four or six codons. Multiple codons for a
single amino acid represent degeneracy in the code.
The genetic code is nearly universal. The same code words are used in all living
organisms, prokaryotic and eukaryotic.
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Codon Usual Code MitochondrialCode
UGA
AUA
AGA
AGG
Termination
Isoleucine
Arginine
Arginine
Tryptophan
Methionine
Termination
Termination
Non universal Codon Usage in Mammalian Mitochondria
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Translation of the codons of mRNA involves their direct interaction with
complementary anticodon sequences in tRNA. Each tRNA species carries a unique amino acid, and each has a specific three-base
anticodon sequence
codon-anticodon base pairing is Antiparallel
codons are read in a sequential, nonoverlapping reading frame
Anticodon and amino acid acceptor sites are located at opposite extremes of all tRNA
molecules
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Variances from standard base pairing are common in the codon-anticodon interactions.
Many amino acids can be carried by more than one tRNA species, and degenerate codons can
be read by more than one tRNA (but always one carrying the correct amino acid).
Wobble - the ability of one tRNA to recognize two or three different mRNA codons .
Occurs when the third base (5end) of the tRNA anticodon has some play or wobble, so that it can
hydrogen bond with more than one kind of a base in the third position (3 end) of the codon.
E.g.: the base U in the wobble position of a tRNA anticodon can pair with either A or G in the third
position of an mRNA codon
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Some tRNAs contain a modified base called inosine (I), which is in the
anticodons wobble position and can pair with U, C, or A in the third position of
an mRNA codon.
Thus, a single tRNA with the anticodonCCI
will recognize three mRNA
codons: GGU, GGC, or GGA all coding for glycine.
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3 Codon Base 5 Anticodon
Bases Possible
Some codons are read more effeciently by one anticodon than another. Not
all codons are used equally, some being used very rarely. Examination of many mRNAsequnces has allowed construction of codon usage tables that show that different
organisms preferentially use different codons to generate similar polypeptide
sequences.
3 Codon Base 5 Anticodon
Bases Possible
A
C
G
U
U or I
G or I
C or U
A or G or I
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The Organization of mRNAs and the Initiation of Translation
UTR - untranslated regions
polycistronic -messenger RNAsthat encode multiple
polypeptide chains
monocistronic -messenger RNAsthat encode a single polypeptide
chain.
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-both prokaryotic and eukaryotic cells, translation always initiates with
the amino acid methionine, usually encoded by AUG.
-In most bacteria, protein synthesis is initiated with a modified methionine
residue (N-formylmethionine), whereas unmodified methionines initiateprotein synthesis in eukaryotes (except in mitochondria and chloroplasts,
whose ribosomes resemble those of bacteria).
Figure 7.7. Signals for translationinitiation
Shine-Delgarno sequence -Thesequence prior to the initiation
site that correctly aligns bacterial
mRNAs on ribosomes.
Signals that identify initiation codons are different in prokaryotic andeukaryotic cells
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Figure 7.8. Overview of translation
The Process of Translation
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Translation cycle
Only two sites are occupied
at any time.
Ribosome is a ribozyme. The crucialfunctions are mostly performed by rRNAs.
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Translation factors
Role Prokaryotes Eukaryotes
Initiation IF-1, IF-2, IF-3 eIF-1, eIF-1A, eIF-2,
eIF-2B, eIF-3, eIF-4A,
eIF-4B, eIF-4E, eIF-4G,
eIF-5
Elongation EF-Tu, EF-Ts, EF-G eEF-1, eEF-1, eEF-
2
Termination RF-1, RF-2, RF-3 eRF-1, eRF-3
Table 7.1. Translation Factors
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Initiation with initiation factors
This ester bond is labile
and protected by eIF2.
In eukaryotes, small subunit
binds to the cap of mRNA and
moves to the first AUG codon.
Eukaryotic mRNA are
monocistronic due to this
initiation mechanism.
Some viral mRNA have an
internal ribosome entrysite (IRES) in the middle.
In bacteria, small subunit
binds to a Shine-Dalgarno
sequence anywhere in mRNA
to initiate translation. Thus,
bacterial mRNA are
polycistronic as they are
produced from an operon.
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It is the association of all of the
eIF4 factors that serves to bring the
mRNA to the 40S subunit..the
eIF4G interacts with the eIF3 on the40s subunit.
Then the 40S subunit with these
factors and tRNA attached scans
the mRNA for the initator (AUG)
codon.
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When the 40S ribosome complex encounters the AUG codon, the eIF5 initiates
hydrolysis of the GTP bound to eIF2.
causes eIF2 which is bound to GTP to
lose a phosphate. This means thateIF2 and any other associated factors
are now only bound to GDP.
This hydrolysis event, signals for
the release of any factor
associated with GDPtheremoval of eIF2, eIF4A, 4, eIF1A,
eIF1, eIF3, eIF4E and eIF4G
This serves as the signal to allow the 60s
Subunit to bind to the 40s Subunit. This isreferred to as the 80s initiation complex.
Once this has formed, then elongation of the protein can begin.
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Bacterial ribosomes engaged in elongating a polypeptide chain exist as 70S
particles.
Initiation of protein synthesis is not a function of intact ribosomes, but is
undertaken by
the separate subunits, which reassociate during the initiation reaction.
Initiation occurs at a special sequence on mRNA called the ribosome-binding site. Thisis a short sequence of bases that precedes the codingregion .The small and large subunits associate at the ribosome binding site
to form an intact ribosome.
The reaction occurs in two steps:
Recognition of mRNA occurs when a small subunit binds to form an
initiation complex at the ribosome-binding site.
A large subunit then joins the complex to generate a complete ribosome.
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Although the 30S subunit is involved in initiation, it is not by itself
competent to undertake the reactions of binding mRNA and tRNA.
It requires additional proteins called initiation factors (IF). These factorsare found only on 30S subunits, and they are released when the 30S subunitsassociate with 50S subunits to generate 70S ribosomes. This behavior
distinguishes initiation factors from the structural proteins of the ribosome.
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Several proteins called elongation factors take part in this three step
cycle which adds amino acids one by one to the initial amino acid:
1. Codon recognition.2. Peptide bond formation.
3. Translocation.
The ribosome as 3 sites for tRNA binding.the P or peptidyl site,
the A or aminoacyl site and E or exit site. The A, E and P sites are used overand over during elongation.
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The elongation cycle of translation - overview
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(Prokaryotes)
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1. Codon recognition
The mRNA codon in the A site of the ribosome forms hydrogen bonds
with the anticodon of an entering tRNA carrying the next amino acid in the
chain.
An elongation factor EF-Tu directs tRNA into the A site in bacteria. In
eukaryotes eEF-1 (4 subunits: eEF-1, eEF-1, eEF-1, eEF-1)
eEF-1 consists of eEF-11 and eEF-12
Hydrolysis of GTP provides energy for this step.
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2. Peptide bond formation.
A peptide bond is formed between the
polypeptide in the P site and the new amino acidin the A site by a peptidyl transferase. Thisreaction requires hydrolysis of GTP bound to EF-Tu, or eEF1.
This inactivates EF-Tu, it is ejected from the
ribosome and regenerated by EF-Ts. Noeukaryotic homology of EF-Ts is known, butpossibly one of the subunits of the eEF-1 has suchactivity.
Peptidyl transferase activity appears to be one
of the rRNAs in the large ribosomal subunit .
The polypeptide separates from its tRNA andis transferred to the new amino acid carried bythe tRNA in the A site.
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3. Translocation.
The tRNA in the A site, which is
now attached to the growing peptide, istranslocated to the P site.Simultaneously, the tRNA that was inthe P site is translocated to the E siteand from there it exits the ribosome.
During this process, the codon and
anticodon remain bonded, so thatmRNA and the tRNA move as a unit,bringing the next codon to betranslated into the A site.
The mRNA is moved through the
ribosome only in the 5 to 3 direction.
Translocation requires GTPhydrolysis and is mediated by EF-G inbacteria and by eEF-2 in eukaryotes.
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3. Translocation.
The tRNA in the A site, which is now attached to the growing
peptide, is translocated to the P site. Simultaneously, the tRNAthat was in the P site is translocated to the E site and from there itexits the ribosome.
During this process, the codon and anticodon remain
bonded, so that mRNA and the tRNA move as a unit, bringingthe next codon to be translated into the A site.
The mRNA is moved through the ribosome only in the 5 to 3direction.
Translocation requires GTP hydrolysis and is mediated by EF-G in bacteria and by eEF-2 in eukaryotes.
This process continues until a termination codon is encountered by the
ribosome.
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Proofreading in elongation
Translation does not need to be as extremely accurate as DNA replication.
Yet, there are several error correction steps in translation
This ester bond is labileand protected by EF-Tu.
All four base-pairs
have identical Hbond
patterns in
the minor grooves.
In all base-pairs,
glycosidic bond
positions are the
same, which is usedin checking correct
base-pairings.
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Termination of Translation
Typical stop codons are UAA, UAG, and UGA. When one of these
sequences is translocated into the A site, this is the signal to stoptranslation.
-Cells do not contain tRNAs with anticodons that are complimentary
to these sequences, so it is impossible for translation to continue.
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There are release factors that recognize this sequence and terminate
protein synthesis.
In eukaryotic cells, there is one release factor (eRF1) that recognizes
all 3 stop codons.
(Prokaryotes)
Termination with release factors
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The release factors
resemble a tRNA to enter
the A site and provides
an H2O molecule tohydrolyze the last tRNA
off the polypeptide
Nascent peptide moves
through a water-filled tunnel.
The walls made of large subunitrRNA are like Teflon coating
for easy sliding. Small
hydrophobic spots are embedded
in extensive hydrophilic surface
of the wall.
Bacterial mRNA aretranslated without
processing before
transcription termination.
Eukaryotic mRNA form a
circle to allow for rapid
re-binding of ribosome.
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What if you need lots of protein to carry out a
particular function within a cell?
-A single mRNA can be translated
simultaneously by several ribosomes in both
prokaryotic and eukaryotic cells.
-Once a ribosome has moved away from the
initaiton site another ribosome can bind and
start synthesis of a new polypeptide chain. This
is what you see here.
-It is common for a single mRNA to be translated
by several ribosomes spaces about 100-200
nucleotides apart. When this occurs and there
are multiple ribosoem attached to the mRNA it
is referred to as a polyribosome.
Each ribosome synthesizes its own separatepolypeptide chain.
-This allows for a much more rapid syntheis of a
given protein. Why might this be important? To
allow a cell to respond to its environment
quickly
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INHIBITOR SPECIFIC EFFECT
Acting only on bacteria
Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome
Streptomycin prevents the transition from initiation complex to chain-elongating
ribosome and also causes miscoding
Chloramphenicol blocks the peptidyl transferase reaction on ribosomes (step 2)
Erythromycin blocks the translocation reaction on ribosomes (step 3)
Rifamycin blocks initiation of RNA chains by binding to RNA polymerase
(prevents RNA synthesis)
Acting on bacteria and eucaryotes
Puromycin causes the premature release of nascent polypeptide chains by its
addition to growing chain end
Actinomycin D binds to DNA and blocks the movement of RNA polymerase (prevents
RNA synthesis)
Acting on eucaryotes but not bacteriaCycloheximide blocks the translocation reaction on ribosomes (step 3)
Anisomycin blocks the peptidyl transferase reaction on ribosomes (step 2 )
-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II
The ribosomes of eucaryotic mitochondria (and chloroplasts) often resemble those of bacteria in their sensitivity to inhibitors Therefore
Table 6-3. Inhibitors of Protein or RNA Synthesis
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