Molecular Biology Fourth Edition

1. Molecular BiologyFourth Edition Chapter 18 The Mechanism of Translation II: Elongation and Termination Chapter 17 The Mechanism of Translation I: Initiation Chapter 19 Ribosomes and Transfer RNA Robert F. Weaver

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 • AUGUUC • No overlapping AUG UUC • Overlapping AUG UGU GUU UUC • If no overlapping, a change of one base in an mRNA would change no more than one a.a. in the resulting protein. • If overlapping, up to three adjacent amino acids could be changed.

6. No-overlapping One base change in TMV mRNA never caused more than one a.a. mutation

7. 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

8. 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

9. Frameshift Mutation Sequences

10. 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

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. How to test the the triplet code hypothesis In 1961, Nirenberg and Matthaei, Poly(U) can be translated into poly-Phe Synthetic mRNA of defined sequence can shed light on the nature of the code

13. First, the codons contained odd number of bases. Poly[UC] or UCUCUCUC……. Odd number of bases------UCU or CUC will code for di-peptide Even number of bases-----CUCU or UCUC will code for single repeat Found…. code for poly(ser-leu)---So, the codons contained an odd number of bases

14. Odd bases! Poly(UC) Three bases? Poly(UUC) Four bases? Poly(UAUC)

15. Four different bases, only twenty amino acids Triplet 4x4x4=64 Two-base codon 4x4=16 not enough combination Nine-base codon 49=262,144 too many

16. The Genetic Code

17. 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 (to reduce the number of tRNA to translate genetic code)

18. Wobble Base Pairs • Compare standard Watson-Crick base pairing with wobble base pairs • Wobble pairs are: • G-U • I-A (or C, or U)

19. Wobble Position

20. 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

21. Deviations from “Universal” Genetic Code

22. 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

23. Elongation in Translation

24. 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

25. Puromycin Structure and Activity

26. 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

27. 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

28. Aminoacyl-tRNA Binding to Ribosome A Site

29. 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

30. 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

31. 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

32. 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

33. Assay for Peptidyl Transferase

34. 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

35. 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

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

37. 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

38. 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

39. 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

40. 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

41. 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

42. Amber Mutation Effects in a Fused Gene

43. 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

44. Termination Mutations

45. 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

46. 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

47. Release Factor Assays

48. 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

49. 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

50. 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