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CHAPTER 6
Gene Expression: Translation
Chapter 5 slide 1
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 3
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).
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Chapter 5 slide 4
Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to chemical type
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 5
Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to chemical type (continued)
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 6
Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to chemical type (continued)
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 7
5. Polypeptides are chains of amino acids joined by covalent peptide bonds. A peptide bond forms between the carboxyl group of one 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.
601 20000 Chapter 5 slide 8
Fig. 6.3 Mechanism for peptide bond formation between the carboxyl group of one amino acid and the amino group of another amino acid
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 9
2. More than amino acid sequence alone determines the folding of a polypeptide into a functional protein. Cell biology experiments show that proteins in the molecular chaperone family assist other proteins in 601 20000 Chapter 5 slide 10 folding.
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 11
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Chapter 5 slide 12
3. The rII mutant strain used in the experiments was produced by treating r+ phage with proflavin. Proflavin causes frameshift mutants by inserting or deleting base pairs of DNA. 4. Crick and colleagues(1961) reasoned that reversion of a deletion (a mutation) could be caused by a nearby insertion (a + mutation) , and vice versa. Revertants of rII to r+ can be detected by plaques on E. coli K12() 5. Combine genetically distinct rII mutants of the same type (either all + or all -), and only when it was a combination of three (or multiple of three) were there high levels of reversion. This indicates that the genetic code is a triplet code.
601 20000 Chapter 5 slide 13
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Chapter 5 slide 14
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 15
Fig. 6.6 Hypothetical example showing how three nearby + (addition) mutations restore the reading frame, giving normal or near-normal function
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 16
c. The amino acid carried by that tRNA corresponds with the codon. About 50 codons were clearly identified using this approach.
4. Both of these techniques were important in understanding the genetic code, and all 61 codons have now been assigned to amino acids; the other three codons do not specify amino acids (Figure 6.7). 5. By convention, a codon is written as it appears in mRNA, reading in the 53 direction.
601 20000 Chapter 5 slide 18
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 19
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 chainterminating 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 6.8).
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Chapter 5 slide 20
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 21
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Chapter 5 slide 22
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.
601 20000 Chapter 5 slide 23
2. When used for in vitro synthesis of hemoglobin, the tRNA inserted alanine at sites where cysteine was expected.
3. The concluded that the specificity of codon recognition lies in the tRNA molecule, and not in the amino acid it carries.
601 20000 Chapter 5 slide 24
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 6.9). 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 3r end of the tRNA. Every tRNA has a 3r adenine, and the amino acid is attached to the 3rOH or 2rOH of this nucleotide.(Figure 6.10).
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Fig. 6.9 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an aminoacyl-tRNA (charged tRNA)
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 26
Fig. 6.10 Molecular details of the attachment of an amino acid to a tRNA molecule
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 27
Initiation of Translation
Animation: Initiation of Translation
1. 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.
4. Prokaryotic translation begins with binding of the 30S ribosomal subunit to mRNA near the AUG codon (Figure 6.11). The 30S comes to the mRNA bound to:
a. All three initiation factors, IF1, IF2 and IF3. b. GTP. c. Mg2+.
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 30
Fig. 6.12 Sequences involved in the binding of ribosomes to the mRNA in the initiation of protein synthesis in prokaryotes
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 31
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 5r-CAU-3r).
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 relased. 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 ribosomes P site.
601 20000 Chapter 5 slide 32
ii. Then the 40S subunit, complexed with initiator Met-tRNA, several eIFs and GTP, binds the cap complex, along with other eIFs.
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 ribosomes P site. c. The eukaryotic mRNAs 3r poly(A) tail also interacts with the 5r 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.
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Chapter 5 slide 33
1. Elongation of the amino acid chain has three steps (Figure 6.13):
a. Binding of aminoacyl-tRNA to the ribosome. b. Formation of a peptide bond. c. Translocation of the ribosome to the next codon.
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Chapter 5 slide 34
Fig. 6.13
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 35
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. When the charged tRNA hydrogen bonds with the codon in the ribosomes 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.
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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. c. The tRNA in the A site now has the growing polypeptide chain attached to it.
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Chapter 5 slide 37
Fig. 6.14 The formation of a peptide bond between the first two amino acids of a polypeptide chain is catalyzed on the ribosome by peptidyl transferase
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 38
Translocation
1. 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 6.15).
601 20000 Chapter 5 slide 39
Fig. 6.15 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.
601 20000
Chapter 5 slide 40
Termination of Translation
Animation: Translation Termination 1. Termination is signaled by a stop codon (UAA, UAG, UGA), which has no corresponding tRNA (Figure 6.16). 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.
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 42
Fig. 6.17x Movement of secretory proteins through the cell membrane system
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 44
Fig. 6.17 Model for the translocation of proteins into the endoplasmic reticulum in eukaryotes
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 45