Chapter 14: The Mechanism of Translation

1. Chapter 14: The Mechanism of Translation

2. We can do experiments on rRNAs today that are far more powerful than anything we ever attempted with ribosomal proteins in the past. So today we work on rRNA; we would be crazy not to. Peter B. Moore, The Ribosome. Structure, Function, and Evolution (1990), p. xxi

3. 14.1 Introduction

4. Ribosomes are assembled in the nucleolus of the cell. • tRNAs are “charged” with their appropriate amino acids. • All the players in protein synthesis join together in the cytoplasm.

5. 14.2 Ribosome structure and assembly

6. Ribosomes are the protein synthesis machinery • Assemble in the nucleolus of the cell and are exported to the cytoplasm. • Decode the genetic code in the mRNA. • Catalyze the formation of peptide bonds between amino acids.

7. “This seems a satisfactory name and it has a pleasant sound.” Dick Roberts (1958, upon proposing the name ribosome)

8. Structure of ribosomes • Determined through a combination of X-ray crystallography, cryoelectron microscopy, biochemical, and genetic data. • Ribosomes consist of two subunits, large and small, composed of ribosomal RNA and many ribosomal proteins.

9. What is “S”? • Ultracentrifugation was developed in the 1920s by Theodor Svedberg. • “S” is one Svedberg unit, which equals a sedimentation coefficient of 10-13 seconds, during density-gradient centrifugation. • S increase with particle mass, but is only a rough estimate of molecular weight.

10. Three-dimensional models of ribosomes • The small subunit includes a head, base, and platform. • The large subunit includes the central protuberance, ridge, and stalk.

11. There are three tRNA binding sites on the ribosome that bridge the large and small subunits • (A) acceptor • (P) peptidyl • (E) exit

12. The peptidyl transferase center is in the large subunit. • Decoding the mRNA occurs on the small subunit.

13. The nucleolus • Non-membrane-bound subcompartment of the nucleus. • Eukaryotic large and small ribosomal subunits are assembled within the nucleolus. • Site of 45S preribosomal RNA transcription by RNA polymerase I.

14. The nucleolus • The 45S pre-rRNA is processed to form the 28S rRNA, 18S rRNA, and 5.8S rRNA. • 5S rRNA is transcribed by RNA pol III. • The 28S, 5.8S, and 5S rRNAs are assembled with ribosomal proteins to form the large subunit. • The 18S rRNA and associated proteins form the small subunit.

15. Ribosome biogenesis • The earliest precursor particle is the 90S precursor, associated with partially processed 35S pre-rRNA. • Cleavage of the 35S pre-rRNA splits the 90S precursor into the 40S and 60S pre-ribosomal subunits. • After export into the cytoplasm via the nuclear pore complexes, the remaining nonribosomal factors dissociate.

16. 14.3 Aminoacyl-tRNA synthetases

17. The overall fidelity of translation is dependent on the accuracy of two processes • Codon-anticodon recognition • Aminoacyl-tRNA synthesis

18. Aminoacyl-tRNA charging • Aminoacyl-tRNA synthetases attach an amino acid to a tRNA in two enzymatic steps: • The amino acid reacts with ATP to become adenylylated (addition of AMP) and pyrophosphate is released. • AMP is released and the amino acid is transferred to the 3′ end of the tRNA.

19. Each aminoacyl-tRNA synthetase is able to precisely match a particular amino acid with the tRNA containing the correct corresponding anticodon. • Specific aminoacyl-tRNA synthetases are denoted by their three-letter amino acid designation. • e.g. MetRS for methionyl-tRNA synthetase

20. Nomenclature • Methionine tRNA or tRNAMet indicates the uncharged tRNA specific for methionine. • Methionyl-tRNA or Met-tRNA denotes the tRNA aminoacylated with methionine.

21. Experimental example • Charging of tRNACUA with pyrrolysine. • A special tRNA called tRNACUA and a novel archael aminoacyltRNA synthetase, PylS are required for incorporation of pyrrolysine.

22. Proofreading activity of aminoacyl-tRNA synthetases • Overall error rate of about one in 10,000 achieved by two means: • Initial high fidelity selection • Proofreading

23. Editing domain of ThrRS • ThrS can distinguish between serine and threonine, despite the similarity in side chains. • Water-mediated hydrolysis of the mischarged tRNA. • The correct product, Thr-tRNAThr is not hydrolyzed due to steric exclusion from the deep editing pocket.

24. 14.4 Initiation of translation

25. Translation can be divided into three main stages • Initiation • Elongation • Termination • Initiation is the most complex and tightly controlled step in protein synthesis.

26. The process of protein synthesis is fundamentally the same in bacteria and eukaryotes. • The difference lies in the details of some of the steps and in the components used to accomplish each step. • Focus on eukaryotic protein synthesis, with reference to bacterial protein synthesis for comparison.

27. Initiation is subdivided into four steps • Ternary complex formation and loading onto the 40S ribosomal subunit. • Loading of the mRNA. • Scanning and start codon recognition. • Joining of the 40S and 60S subunits to form the functional 80S ribosomes.

28. Ternary complex formation and loading onto the 40S ribosomal subunit • The ternary complex is composed of: • Eukaryotic initiation factor 2 (eIF2) • GTP • The amino acid-charged initiator tRNA (Met-tRNA) • The ternary complex binds the 40S ribosomal subunit, plus other initiation factors, including eIF4G/E, to form a 43S complex.

29. Loading the mRNA on the 40S ribosomal subunit • eIF4G and eIF4E, initiation factors with RNA helicase activity, associate with the 5′ cap of the mRNA and unwind any secondary and tertiary structures. • Other initiation factors associate with the poly(A)-binding protein (PABP) bound to the 3′-poly(A) tail.

30. The closed-loop model of translation initiation • The 5′-cap and 3′-poly(A) tail of the mRNA join to form a closed loop with eIF4G serving as the bridge between them. • Some cellular RNAs are translated by a 5′-cap-independent mechanism in which ribosomes are directly recruited by an internal ribosome entry site (IRES).

31. Scanning and start codon recognition • Once the mRNA is loaded, the 43S complex scans along the message from 5′→3′ looking for the AUG start codon. • ATP-dependent mechanism. • AUG is embedded in a Kozak consensus sequence.

32. Translation toeprinting assays • mRNA is translated using ribosomal components. • The mRNA complex is copied into cDNA by reverse transcriptase using a complementary labeled primer. • Where the reverse transcriptase meets the ribosome bound to the mRNA, polymerization is halted and a “toeprint” cDNA fragment is generated.

33. Toeprinting assays have been used to characterize the initiation factors required for AUG recognition in either a “good” or “bad” sequence context.

34. Joining of the 40S and 60S ribosomal subunits to form the functional 80S ribosomes • Initiation factors are released from the 43S complex in a process that requires GTP hydrolysis. • eIF2-GDP is converted to eIF2-GTP through a nucleotide exchange reaction mediated by eIF2B.

35. The 60S subunit joins with the 40S subunit to form the 80S ribosome initiation complex, in a process that requires a second GTP hydrolysis step. • eIF5-GDP is converted to eIF5-GTP through a nucleotide exchange reaction mediated by eIF5B.

36. Eukaryotic initiation factor 2B and vanishing white matter • Recessively inherited, fatal disease. • Progressive loss of movement and speech, seizures, and coma usually before age 5. • Episodes of rapid and major deterioration following fever or minor head trauma.

37. White matter (myelinated axons) vanishes and is replaced by cerebrospinal fluid. • Mutations in the genes encoding the 5 subunits of guanine nucleotide exchange factor eIF2B. • Why the cells that make myelin are particularly sensitive to defects in eIF2B remains unknown.

38. 14.5 Elongation and events in the ribosome tunnel

39. Peptide chain elongation begins with a peptidyl-tRNA in the ribosomal P site next to a vacant A site. • An aminoacyl-tRNA is carried to the A site as part of a ternary complex with GTP-bound eEF1A.

40. Upon GTP hydrolysis, the aminoacyl-tRNA enters the A site where decoding takes place. • eEF1A-GDP is converted to eEF1A-GTP through a nucleotide exchange reaction mediated by eEF1B.

41. The incorporation of selenocysteine at a specific UGA site depends upon: • tRNASel • Elongation factor SelB and SBP2 (in eukaryotes) • A selenocysteine insertion sequence (SECIS) • Pyrrolysine insertion elements (PYLIS) in archaea signal for pyrrolysine incorporation at a UAG codon.

42. Decoding the message • tRNA selection involves two steps: initial selection and proofreading. • The two steps are separated by irreversible hydroylsis of GTP. • When the ternary complex contains the correct tRNA, the initial selection step occurs more rapidly and GTP hydrolysis releases the tRNA in the A site.

43. Conformational changes in tRNA are the basis for induced fit, which is essential for high-fidelity tRNA selection. • Only one incorrect amino acid per 1000 to 10,000 correct amino acids.

44. Experimental evidence for the importance of tRNA conformation in decoding • Studies of a mutant called the “Hirsch suppressor,” a tRNATrp variant that has a single G24A substitution in the D arm. • Mutant tRNA recognizes both UGG and UGA stop codons even though the mutation does not alter the anticodon.

45. The Hirsch suppressor mutant accelerates the forward rate constants during decoding independent of codon-anticodon pairing.

46. Peptide bond formation and translocation • Peptidyl transferase activity transfers a growing polypeptide chain from peptidyl-tRNA in the P site to an amino acid esterified with another tRNA in the A site. • After the tRNAs and mRNA are translocated and the next codon is moved to the A site, the process is repeated. • Mediated by eEF2; requires GTP hydrolysis.

47. Peptidyl transferase activity • The site of peptide bond formation is located at the base of the central protuberance in the large ribosomal subunit. • A central question for many years was whether the “peptidyl transferase activity” that catalyzes peptide bond formation is the result of a protein or RNA enzyme.

48. Biochemical evidence that 23S rRNA is a ribozyme • “Fragment reaction” used by Harry Noller and colleagues to shown that purified bacterial 23S rRNA has “peptidyl transferase activity” in vitro.

49. Structural evidence that rRNA forms the active site of the ribosome • X-ray crystallographic images at atomic (2.4Å) resolution of archeon Haloarcula marismortui large ribsosomal subunits. • rRNA forms the catalytic center, decoding site, A, P and E sites, and the intersubunit interface. • Ribosomal proteins are abundant on the exterior of the ribosome.

50. The ribosome is a ribozyme • The peptidyl transferase center is located in domain V of the 23S rRNA. • One model proposes that the 107-fold rate of enhancement of peptide bond formation is due to substrate positioning by the 23S rRNA within the active site, rather than chemical catalysis.