chapter 6 gene expression: translationanisam2.yolasite.com/resources/26nov10llecture16.pdf ·...

台大農藝系遺傳學 601 20000 Chapter 5 slide 1

Gene Expression: Translation

transmission of information from mRNA to proteins

台大農藝系遺傳學 601 20000 Chapter 5 slide 2Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.1 General structural formula for an amino acid


4. There are 20 amino acids used in biological proteins. They are divided into subgroups according to the properties of their R groups (acidic, basic, neutral and polar, or neutral and nonpolar) (Figure 6.2).


Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to chemical type


Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to chemical type (continued)


5. Polypeptides are chains of amino acids joined by covalent peptide bonds. A peptide bond forms between the carboxyl group of 1 amino acid, and the amino group of another (Figure 6.3).

6. Polypeptides are unbranched, and have a free amino group at one end (the N terminus) and a carboxyl group at the other (the C terminus). The N-terminal end defines the beginning of the polypeptide.


Fig. 6.3 Mechanism for peptide bond formation between the carboxyl group of one amino acid and the amino group of another amino acid


Translation: The Process of Protein Synthesis1. Ribosomes translate the genetic message of mRNA into

proteins.

2. The mRNA is translated 5’3’, producing a corresponding N-terminal C-terminal polypeptide.

3. Amino acids bound to tRNAs are inserted in the proper sequence due to:a. Specific binding of each amino acid to its tRNA.

b. Specific base pairing between the mRNA codon and tRNA anticodon.


Deciphering/Decoding the Genetic Code

1. The relationship between codons and amino acids was determined by Nirenberg and Khorana (1968) using cell- free, protein-synthesizing systems from E. coli that included ribosomes and required protein factors, along with tRNAs carrying radiolabeled amino acids.

2. There are 64 possible codons (4 x 4 x 4). All 61 codons have now been assigned to amino acids; the other three codons do not specify amino acids (Figure 6.8).

2. By convention, a codon is written as it appears in mRNA, reading in the 5’3’ direction.

Marshall Nirenberg HarGobind Khorana


Fig. 6.8 The genetic code


Characteristics of the Genetic Code1. Characteristics of the genetic code:

a. It is a triplet code. Each three-nucleotide codon in the mRNA specifies 1 amino in the polypeptide.

b. It is comma free. The mRNA is read continuously, three bases at a time, without skipping any bases.

c. It is non-overlapping. Each nucleotide is part of only one codon, and is read only once during translation.

d. It is almost universal. In nearly all organisms studied, most codons have the same amino acid meaning. Examples of minor code differences include the protozoan Tetrahymena and mitochondria of some organisms.

e. It is degenerate. Of 20 amino acids, 18 are encoded by more than one codon. Met (AUG) and Trp (UGG) are the exceptions; all other amino acids correspond to a set of two or more codons. Codon sets often show a pattern in their sequences; variation at the third position is most common (Figure 6.8).

f. The code has start and stop signals. AUG is the usual start signal for protein synthesis. Stop signals are codons with no corresponding tRNA, the nonsense or chain- terminating codons. There are generally three stop codons: UAG (amber), UAA (ochre) and UGA (opal).

g. Wobble occurs in the anticodon. The 3rd base in the codon is able to base-pair less specifically, because it is less constrained three-dimensionally. It wobbles, allowing a tRNA with base modification of its anticodon (e.g., the purine inosine) to recognize up to three different codons (Figure 6.9).


Fig. 6.9 Example of base-pairing wobble


• Translation rate: 2-20 amino acids per second (compared to DNA synth. 200-1000 bp/sec)

• Ribosomes have no proofreading activity.

• AminoacyltRNA synthetases have editing function.


Translation is highly conserved in prokaryotes and eukaryotes• The major events are very similar

• The main differences are in the initiation process with related effects on mRNA structure

• Prokaryotes have polycistronic mRNA, with internal initiation by ribosomes

• Eukaryotes have monocistronic mRNA, with initiation only at 5’ end cap structures

• Prokaryotes couple transcription and translation in the same cellular compartment

• Eukaryotes separate transcription (nuclear) from translation (cytoplasmic)


The Initiation of Translation in Pro- and Eukaryotic Cells

5’ cap: After 20-30 nucleotides have been synthesized, the 5’- end of the mRNA is capped 5’ to 5’ with a guanine nucleotide. Essential for the ribosome to bind to the 5’ end of the mRNA.

Poly (A) tail: 50-250 adenine nucleotides are added to 3’ end of mRNA. Stabilizes the mRNA, and plays an important role in transcription termination.


mRNAs are translated into proteins

• The role of mRNA is to transfer information from the gene to the protein (gene product)

• mRNAs are translated into proteins by ribosomes

• Translation is a multistep process involving the ribosome and many other enzymes and factors

• Mainly 4 components: mRNA, tRNA, ribosomes, aminoacyl tRNA synthetases.


Eu- and Prokaryotic Ribosomes

Eukaryotic cytoplasm

Prokaryotes, Eukaryotic organelles (mitochondria, chloroplasts)


E: Exit site for free tRNA P: peptidyl-tRNA A: aminoacyl-tRNA

E, P and A Sites of Ribosomes

3 binding sites for tRNAChannels/TUNNELS through the ribosome allow the mRNA and growingPolypeptide to enter and/exit the ribosome


Features of mRNA for translation

• The mRNA Codon Recognizes the tRNA Anticodon

• ORF: read as string of codons (set of 3 nucleotides).

• Start codon: AUG (rarely GUG, UUG in bacteria)

• Stop codon: UAA, UAG, UGA.

• 3 possible reading frames but AUG decides reading frame

• Prok.: have ribosome binding sites (RBS) upstream of start codon called Shine-Dalgarno sequence.


No involvement of mRNA 5’ end

Shine – Dalgarno sequences +AUG initiation codons can occur within 5’ non-translated regions, and, may also occur within site(s) internal to the mRNA …….


Prokaryotic mRNAs may be polycistronic

The ability to bind ribosomes and initiate translation at sites internal to the prokaryotic mRNA allows • genes to be organised into operons, • an operon to be transcribed into a single (polycistronic) mRNA, • the expression of a number of genes (related functions) to be controlledby a single promoter (or single translational control mechanism)

cistron 1 cistron 2cistron 3

sites of ribosome ‘re-cycling’


Initiation of Translation in Eukaryotes

major differences to prokaryotic mRNA……

• eukaryotic mRNAs possess a different 5’ ‘cap’ structure: recruits ribosomes• eukaryotic mRNAs are polyadenylated

Bases around the initiating AUG influence the efficiency of initiation: RNNNAUGG (‘Kozak consensus’ sequence); R = Purine; G next to AUG.

Eukaryotic translation initiation factor eIF4 binds to cap; recruits ribosome and scans along mRNA to find initiator AUG

Kozak consensus’ sequence


Charging tRNA1. Aminoacyl-tRNA synthetase attaches amino acids to their specific

tRNA molecules. The charging process (aminoacylation) produces a charged tRNA (aminoacyl-tRNA), using energy from ATP hydrolysis.

2. There are 20 different aminoacyl-tRNA synthetase enzymes, one for each amino acid. Some of these enzymes recognize tRNAs by their anticodon regions, and others by sequences elsewhere in the tRNA.

3. The amino acid and ATP bind to the specific aminoacyl-tRNA synthetase enzyme. ATP loses two phosphates and the resulting AMP is bound to the amino acid, forming aminoacyl-AMP (Figure 6.10).

4. The tRNA binds to the enzyme, and the amino acid is transferred onto it, displacing the AMP. The aminoacyl-tRNA is released from the enzyme.

5. The amino acid is now covalently attached by its carboxyl group to the 3’ end of the tRNA. Every tRNA has a 3’ adenine, and the amino acid is attached to the 3’–OH or 2’–OH of this nucleotide. (Figure 6.11).


Fig. 6.10 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an aminoacyl-tRNA (charged tRNA)


Fig. 6.11 Molecular details of the attachment of an amino acid to a tRNA molecule


Initiation of Translation1. Protein synthesis is similar in prokaryotes and eukaryotes.

Some significant differences do occur, and are noted below.2. In both it is divided into three stages:

a. Initiation.b. Elongation.c. Termination.

3. Initiation of translation requires:a. An mRNA.b. A ribosome.c. A specific initiator tRNA.d. Initiation factors.e. Mg2+ (magnesium ions).


4. Prokaryotic translation begins with binding of the 30S ribosomal subunit to mRNA near the AUG codon (Figure 6.12). The 30S comes to the mRNA bound to:

a. All three initiation factors, IF1, IF2 and IF3.

IF1: Prevents tRNA from binding to A site

IF2: GTPase; binds GTP; interacts with initiator tRNA, IF1, and small 30S subunit.

IF3: Prevents 30S-50S subunit association.

b. GTP.

c. Mg2+.


Fig. 6.12 Initiation of protein synthesis in prokaryotes


6. Next, the initiator tRNA binds the AUG to which the 30S subunit is bound. AUG universally encodes methionine. Newly made proteins begin with Met, which is often subsequently removed.a. Initiator methionine in prokaryotes is formylmethionine (fMet). It is

carried by a specific tRNA (with the anticodon 5’r-CAU-3’r).

b. The tRNA first binds a methionine, and then transformylase attaches a formyl group to the methionine, making fMet-tRNA.fMET (a charged initiator tRNA).

c. Methionines at sites other than the beginning of a polypeptide are inserted by tRNA.Met (a different tRNA), which is charged by the same aminoacyl-tRNA synthetase as tRNA.fMet.

7. When Met-tRNA.fMet binds the 30S-mRNA complex, IF3 is released and the 50S ribosomal subunit binds the complex. GTP is hydrolysed, and IF1 and IF2 are released. The result is a 70S initiation complex consisting of (Figure 6.14):a. mRNA.

b. 70S ribosome (30S and 50S subunits) with a vacant A site.

c. fMet-tRNA in the ribosome’s P site.


8. The main differences in eukaryotic translation are:

a. Initiator methionine is not modified. As in prokaryotes, it is attached to a special tRNA.

b. Ribosome binding involves the 5’ cap, rather than a Shine-Dalgarno sequence.

i. Eukaryotic initiator factor (eIF-4F) is a multimer of proteins, including the cap binding protein (CBP), binds the 5’ mRNA cap.

ii. Then the 40S subunit, complexed with initiator Met-tRNA, several eIFs and GTP, binds the cap complex, along with other eIFs (total 5 of them).

iii. The initiator complex scans the mRNA for a Kozak sequence that includes the AUG start codon. This is usually the 1st AUG in the transcript.

iv. When the start codon is located, 40S binds, and then 60S binds, displacing the eIFs and creating the 80S initiation complex with initiator Met-tRNA in the ribosome’s P site.

c. The eukaryotic mRNA’s 3’r poly(A) tail also interacts with the 5’r cap. Poly(A) binding protein (PABP) binds the poly(A), and also binds a protein in eIF-4F on the cap, circularizing the mRNA and stimulating translation.


Elongation of the Polypeptide Chain

1. Elongation of the amino acid chain has three steps (Figure 6.15):a. Binding of aminoacyl-tRNA to the ribosome.

b. Formation of a peptide bond.

c. Translocation of the ribosome to the next codon.


Fig. 6.15 Elongation stage of translation in prokaryotes


Binding of Aminoacyl-tRNA

1. Protein synthesis begins with fMet-tRNA in the P site of the ribosome. The next charged tRNA approaches the ribosome bound to EF-Tu-GTP (eEF1 in eukaryotes). When the charged tRNA hydrogen bonds with the codon in the ribosome’s A site, hydrolysis of GTP releases EF-Tu-GDP.

2. EF-Tu is recycled with assistance from EF-Ts, which removes the GDP and replaces it with GTP, preparing EF-Tu-GTP to escort another aminoacyl tRNA to the ribosome.


Peptide Bond Formation

1. The two aminoacyl-tRNAs are positioned by the ribosome for peptide bond formation, which occurs in two steps:a. In the P site, the bond between the amino acid and its tRNA is

cleaved.

b. Peptidyl transferase forms a peptide bond between the now-free amino acid in the P site and the amino acid attached to the tRNA in the A site. Experiments indicate that the 23S rRNA is most likely the catalyst for peptide bond formation; suggests that RIBOSOME IS A RIBOZYME??

c. The tRNA in the A site now has the growing polypeptide chain attached to it.


Translocation1. The ribosome now advances one codon along the mRNA. EF-G is used in

translocation in prokaryotes. EF-G-GTP binds the ribosome, GTP is hydrolyzed and the ribosome moves 1 codon while the uncharged tRNA leaves the P site. Eukaryotes use a similar process, with a factor called eEF-2.

2. Release of the uncharged tRNA involves the 50S ribosomal E (for Exit) site. Binding of a charged tRNA in the A site is blocked until the spent tRNA is released from the E site.

3. During translocation the peptidyl-tRNA remains attached to its codon, but is transferred from the ribosomal A site to the P site by an unknown mechanism.

4. The vacant A site now contains a new codon, and an aminoacyl-tRNA with the correct anticodon can enter and bind. The process repeats until a stop codon is reached.

5. Elongation and translocation are similar in eukaryotes, except for differences in number and type of elongation factors and the exact sequence of events.

6. In both prokaryotes and eukaryotes, simultaneous translation occurs. New ribosomes may initiate as soon as the previous ribosome has moved away from the initiation site, creating a polyribosome (polysome); an average mRNA might have 8-10 ribosomes (Figure 6.17).


Fig. 6.17 Diagram of a polysome, a number of ribosomes each translating the same mRNA sequentially

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.


Termination of Translation1. Termination is signaled by a stop codon (UAA, UAG, UGA), which has no

corresponding tRNA (Figure 6.18).

2. Release factors (RF) assist the ribosome in recognizing the stop codon and terminating translation.

a. In E. coli:

i. RF1 recognizes UAA and UAG.

ii. RF2 recognizes UAA and UGA.

iii. RF3 stimulates termination.

b. In eukaryotes, there is only one termination factor, eRF.

3. Termination events triggered by release factors are:

a. Peptidyl transferase releases the polypeptide from the tRNA in the ribosomal P site.

b. The tRNA is released from the ribosome.

c. The two ribosomal subunits and RF dissociate from the mRNA.

d. The initiator amino acid (fMet or Met) is usually cleaved from the polypeptide.

ENERGY USE: 2 GTP and 1 ATP consumed per peptide bond formation.


Fig. 6.18 Termination of translation

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.


The main advantage of translational control is the speed of the effect on protein levels.

Under stress conditions translation is often inhibited.

Translation can also be activated allowing greater growth and proliferation upon stimulation by hormones, growth factors, mitogens and cytokines.


How do we assess whether individual mRNAsare regulated at the translational level?

glucose to glucose(control)

Western Blotmeasuring Protein levels

Northern Blotmeasuring mRNA levels

glucose to glycerol

glucose to glucose(control)

glucose to glycerol

Example:-In yeast (S. cerevisiae) the YL250c gene


• Translation of specific mRNAs can be increased or decreased by 5’or 3’UTR cis acting sequences.

• These translational controls play important roles in :-

Developmental biology e.g. embryonic development

Virology e.g. foot and mouth disease virus

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