Chapter 18 The Mechanism of Translation II: Elongation and Termination

1. Chapter 18 The Mechanism of Translation II: Elongation and Termination

2. 18.1 Direction of Polypeptide Synthesis and mRNA Translation • Messenger RNAs are read in the 5’3’ direction • This is the same direction in which they are synthesized • Proteins are made in the aminocarboxyl direction • This means that the amino terminal amino acid is added first

3. Strategy to Determine Direction of Translation

4. 18.2 The Genetic Code • The term genetic code refers to the set of 3-base code words (codons) in mRNA that represent the 20 amino acids in proteins • Basic questions were answered about translation in the process of “breaking” the genetic code

5. Nonoverlapping Codons • Each base is part of at most one codon in nonoverlapping codons • In an overlapping code, one base may be part of two or even three codones

6. No Gaps in the Code • If the code contained untranslated gaps or “commas”, mutations adding or subtracting a base from the message might change a few codons • Would still expect ribosome to be back “on track” after the next such comma • Mutations might frequently be lethal • Many cases of mutations should occur just before a comma and have little, if any, effect

7. Frameshift Mutations Frameshift mutations • Translation starts AUGCAGCCAACG • Insert an extra base AUXGCAGCCAACG • Extra base changes not only the codon in which is appears, but every codon from that point on • The reading frame has shifted one base to the left Code with commas • Each codon is flanked by one or more untranslated bases • Commas would serve to set off each codon so that ribosomes recognize it • Translation starts AUGZCAGZCCAZACGZ • Insert an extra base AUXGZCAGZCCAZACGZ • First codon wrong, all others separated by Z, translated normally

8. Frameshift Mutation Sequences

9. The Triplet Code • The genetic code is a set of three-base code words, or codons • In mRNA, codons instruct the ribosome to incorporate specific amino acids into a polypeptide • Code is nonoverlapping • Each base is part of only one codon • Devoid of gaps or commas • Each base in the coding region of an mRNA is part of a codon

10. Coding Properties of Synthetic mRNAs

11. Breaking the Code • The genetic code was broken • Using: • Synthetic messengers • Synthetic trinucleotides • Then observing: • Polypeptides synthesized • Aminoacyl-tRNAs bound to ribosomes • There are 64 codons • 3 are stop signals • Remainder code for amino acids • The genetic code is highly degenerate

12. The Genetic Code

13. Unusual Base Pairs Between Codon and Anticodon Degeneracy of genetic code is accommodated by: • Isoaccepting species of tRNA: bind same amino acid, but recognize different codons • Wobble, the 3rd base of a codon is allowed to move slightly from its normal position to form a non-Watson-Crick base pair with the anticodon • Wobble allows same aminoacyl-tRNA to pair with more than one codon

14. Wobble Base Pairs • Compare standard Watson-Crick base pairing with wobble base pairs • Wobble pairs are: • G-U • I-A

15. Wobble Position

16. Almost Universal Code • Genetic code is NOT strictly universal • Certain eukaryotic nuclei and mitochondria along with at least one bacterium • Codons cause termination in standard genetic code can code for amino acids Trp, Glu • Mitochondrial genomes and nuclei of at least one yeast have sense of codon changed from one amino acid to another • Deviant codes are still closely related to standard one from which they evolved • Genetic code a frozen accident or the product of evolution • Ability to cope with mutations evolution

17. Deviations from “Universal” Genetic Code

18. 18.3 The Elongation Mechanism Elongation takes place in three steps: • EF-Tu with GTP binds aminoacyl-tRNA to the ribosomal A site • Peptidyl transferase forms a peptide bond between peptide in P site and newly arrived aminoacyl-tRNA in the A site Lengthens peptide by one amino acid and shifts it to the A site • EF-G with GTP translocates the growing peptidyl-tRNA with its mRNA codon to the P site

19. Elongation in Translation

20. A Three-Site Model of the Ribosome • Puromycin • Resembles an aminoacyl-tRNA • Can bind to the A site • Couple with the peptide in the P site • Release it as peptidyl puromycin • If peptidyl-tRNA is in the A site, puromycin will not bind to ribosome, peptide will not be released • Two sites are defined on the ribosome: • Puromycin-reactive site (P) • Puromycin unreactive site (A) • 3rd site (E) for deacylated tRNA bind to E site as exits ribosome

21. Puromycin Structure and Activity

22. Protein Factors and Peptide Bond Formation • One factor is T, transfer • It transfers aminoacyl-tRNAs to the ribosome • Actually 2 different proteins • Tu, u stands for unstable • Ts, s stands for stable • Second factor is G, GTPase activity • Factors EF-Tu and EF-Ts are involved in the first elongation step • Factor EF-g participates in the third step

23. Elongation Step 1 Binding aminoacyl-tRNA to A site of ribosome • Ternary complex formed from: • EF-Tu • Aminoacyl-tRNA • GTP • Delivers aminoacyl-tRNA to ribosome A site without hydrolysis of GTP • Next step: • EF-Tu hydrolyzes GTP • Ribosome-dependent GTPase activity • EF-Tu-GDP complex dissociates from ribosome • Addition of aminoacyl-tRNA reconstitutes ternary complex for another round of translation elongation

24. Aminoacyl-tRNA Binding to Ribosome A Site

25. Proofreading • Protein synthesis accuracy comes from charging tRNAs with correct amino acids • Proofreading is correcting translation by rejecting an incorrect aminoacyl-tRNA before it can donate its amino acid • Protein-synthesizing machinery achieves accuracy during elongation in two steps

26. Protein-Synthesizing Machinery • Two steps achieve accuracy: • Gets rid of ternary complexes bearing wrong aminoacyl-tRNA before GTP hydrolysis • If this screen fails, still eliminate incorrect aminoacyl-tRNA in the proofreading step before wrong amino acid is incorporated into growing protein chain • Steps rely on weakness of incorrect codon-anticodon base pairing to ensure dissociation occurs more rapidly than either GTP hydrolysis or peptide bond formation

27. Proofreading Balance • Balance between speed and accuracy of translation is delicate • If peptide bond formation goes too fast • Incorrect aminoacyl-tRNAs do not have enough time to leave the ribosome • Incorrect amino acids are incorporated into proteins • If translation goes too slowly • Proteins are not made fast enough for the organism to grow successfully • Actual error rate, ~0.01% per amino acid is a good balance between speed and accuracy

28. Elongation Step 2 • One the initiation factors and EF-Tu have done their jobs, the ribosome has fMet-tRNA in the P site and aminoacyl-tRNA in the A site • Now form the first peptide bond • No new elongation factors participate in this event • Ribosome contains the enzymatic activity, peptidyl transferase, that forms peptide bond

29. Assay for Peptidyl Transferase

30. Peptide Bond Formation • The peptidyl transferase resides on the 50S ribosomal particle • Minimum components necessary for activity are 23S rRNA and proteins L2 and L3 • 23S rRNA is at the catalytic center of peptidyl transferase

31. Elongation Step 3 • When peptidyl transferase has worked: • Ribosome has peptidyl-tRNA in the A site • Deacylated tRNA in the P site • Translocation, next step, moves mRNA and peptidyl-tRNA one codon’s length through the ribosome • Places peptidyl-tRNA in the P site • Ejects the deacylated tRNA • Process requires elongation factor EF-G which hydrolyzes GTP after translocation is complete

32. Three-Nucleotide Movement Each translocation event moves the mRNA on codon length, or 3 nt through the ribosome

33. Role of GTP and EF-G • GTP and EF-G are necessary for translocation • Translocation activity appears to be inherent in the ribosome • This activity can be expressed without EF-G and GTP • GTP hydrolysis • Precedes translocation • Significantly accelerate translocation • New round of elongation occurs if: • EF-G must be released from the ribosome • Release depends on GTP hydrolysis

34. GTPases and Translation • Some translation factors harness GTP energy to catalyze molecular motions • These factors belong to a large class of G proteins • Activated by GTP • Have intrinsic GTPase activity activated by an external factor (GAP) • Inactivated when they cleave their own GTP to GDP • Reactivated by another external factor (guanine nucleotide exchange protein) that replaces GDP with GTP

35. G Protein Features • Bind GTP and GDP • Cycle among 3 conformational states • Depends on whether bound to: • GDP • GTP • Neither • Conformational state determine activity • Activated to carry out functionality when bound to GTP • Intrinsic GTPase activity

36. More G Protein Features • GTPase activity stimulated by GTPase activator protein (GAP) • When GAP stimulates GTPase cleave GTP to GDP • Results in self inactivation • Reactivation by guanine nucleotide exchange protein • Removes GDP from inactive G protein • Allows another molecule of GTP to bind • Example of guanine nucleotide exchange protein is EF-Ts

37. Structures of EF-Tu and EF-G • Three-dimensional shapes determined by x-ray crystallography: • EF-Tu-tRNA-GDPNP ternary complex • EF-G-GDP binary complex • As predicted, the shapes are very similar

38. 18.4 Termination • Elongation cycle repeats over and over • Adds amino acids one at a time • Grows the polypeptide product • Finally ribosome encounters a stop codon • Stop codon signals time for last step • Translation last step is termination

39. Termination Codons • Three codons are the natural stop signals at the ends of coding regions in mRNA • UAG • UAA • UGA • Mutations can create termination codons within an mRNA causing premature termination of translation • Amber mutation creates UAG • Ochre mutation creates UAA • Opal mutation creates UGA

40. Amber Mutation Effects in a Fused Gene

41. Termination Mutations • Amber mutations are caused by mutagens that give rise to missense mutations • Ochre and opal mutations do not respond to the same suppressors as do the amber mutations • Ochre mutations have their own suppressors • Opal mutations also have unique suppressors

42. Termination Mutations

43. Stop Codon Suppression • Most suppressor tRNAs have altered anticodons: • Recognize stop codons • Prevent termination by inserting an amino acid • Allow ribosome to move on to the next codon

44. Release Factors • Prokaryotic translation termination is mediated by 3 factors: • RF1 recognizes UAA and UAG • RF2 recognizes UAA and UGA • RF3 is a GTP-binding protein facilitating binding of RF1 and RF2 to the ribosome • Eukaryotes has 2 release factors: • eRF1 recognizes all 3 termination codons • eRF3 is a ribosome-dependent GTPase helping eRF1 release the finished polypeptide

45. Release Factor Assays

46. Dealing with Aberrant Termination • Two kinds of aberrant mRNAs can lead to aberrant termination • Nonsense mutations can occur that cause premature termination • Some mRNAs (non-stop mRNAs) lack termination codons • Synthesis of mRNA was aborted upstream of termination codon • Ribosomes translate through non-stop mRNAs and then stall • Both events cause problems in the cell yielding incomplete proteins with adverse effects on the cell • Stalled ribosomes out of action • Unable to participate in further protein synthesis

47. Non-Stop mRNAs • Prokaryotes deal with non-stop mRNAs by tmRNA-mediated ribosome rescue • Alanyl-tmRNA resembles alanyl-tRNA • Binds to vacant A site of a ribosome stalled on a non-stop mRNA • Donates its alanine to the stalled polypeptide • Ribosome shifts to translating an ORF on the tmRNA (transfer-messenger RNA) • Adds another 9 amino acids to the polypeptide before terminating • Extra amino acids target the polypeptide for destruction • Nuclease destroys non-stop mRNA

48. Non-Stop mRNAs • Prokaryotes deal with non-stop mRNAs by tmRNA-mediated ribosome rescue • tmRNA are about 300 nt long • 5’- and 3’-ends come together to form a tRNA-like domain (TLD) resembling a tRNA

49. Eukaryotic Aberrant Termination • Eukaryotes do not have tmRNA • Eukaryotic ribosomes stalled at the end of the poly(A) tail contain 0 – 3 nt of poly(A) tail • This stalled ribosome state is recognized by carboxyl-terminal domain of a protein called Ski7p • Ski7p also associates tightly with cytoplasmic exosome, cousin of nuclear exosome • Non-stop mRNA recruit Ski7p-exosome complex to the vacant A site • Ski complex is recruited to the A site • Exosome, positioned just at the end of non-stop mRNA, degrades that RNA • Aberrant polypeptide is presumably destroyed

50. Exosome-Mediated Degradation • This stalled ribosome state is recognized by carboxyl-terminal domain of a protein called Ski7p • Ski7p also associates tightly with cytoplasmic exosome, cousin of nuclear exosome • Non-stop mRNA recruit Ski7p-exosome complex to the vacant A site • Ski complex is recruited to the A site