Ribosome Functions and Proportions in Translation in Prokaryotes & Eukaryotes

1. Translation in prokaryotes & eukaryotes CA García Sepúlveda MD PhD Laboratorio de Genómica Viral y HumanaFacultad de Medicina, Universidad Autónoma de San Luis Potosí

2. Introduction • Ribosomes provide the environment for controlling the interaction between mRNA and aminoacyl-tRNA. • Behave like a small migrating factories travelling along the mRNA template engaging in rapid cycles of peptide bond synthesis. • Possess several active centers, each constructed from a particular group of proteins associated with rRNA. • The active centers require rRNA for structural and catalytic roles. • Some catalytic functions require particular proteins, but none of the activities can be reproduced by isolated proteins or groups of proteins; they function only in the context of the ribosome (associated with RNA).

3. Molecular Proportions • Each ribosome subunit has specific roles. • mRNA is associated with the small subunit (~30 bases of the mRNA). • tRNAs are quite large relative to the ribosome; they become inserted into internal sites that stretch across the subunits. • A third tRNA may remain present on the ribosome after it has been used in protein synthesis, before being recycled (prokaryotes only). • Polypeptide elongation involves reactions at just two of the ~10 codons covered by the ribosome.

4. Ribosomes • The basic form of the ribosome is conserved, but there are appreciable variations in the overall size and proportions of RNA and protein in the ribosomes of bacteria, eukaryotic cytoplasm, and organelles. Al ribosomes consists of two subunits, each of which contains a major rRNA and a number of small proteins. The large subunitmay also contain smaller RNAs.

5. Overview • Each tRNA lies in a distinct site on the ribosome, the two sites have different features: • Incoming aminoacyl-tRNA binds to the codon exposed on the A site (or acceptor site).

6. Overview • Each tRNA lies in a distinct site on the ribosome, the two sites have different features: • A salient peptidyl-tRNA (tRNA carrying the polypeptide) occupies the codon in the P site (or donor site).

7. Overview • Each tRNA lies in a distinct site on the ribosome, the two sites have different features: • The anticodon at the other end interacts with the mRNA bound by the small subunit. • So the P and A sites each extend across both ribosomal subunits.

8. Overview • Each tRNA lies in a distinct site on the ribosome, the two sites have different features: • Peptide bond formation occurs when the polypeptide carried by the peptidyl-tRNA is transferred to the amino acid carried by the aminoacyl-tRNA. • This reaction is catalyzed by constituents of the large subunit of the ribosome.

9. Overview • Transfer of the polypeptide generates the ribosome shown in step 2. • The deacylated tRNA, lacking any amino acid, lies in the P site, while a new peptidyl-tRNA has been created in the A site. • This peptidyl-tRNA is one amino acid residue longer than the peptidyl-tRNA that had been in the P site in step 1.

10. Overview • Then the ribosome moves one triplet along the messenger = TRANSLOCATION.

11. Overview • Then the ribosome moves one triplet along the messenger = TRANSLOCATION. • The movement transfers the deacylated tRNA out of the P site...

12. Overview • Then the ribosome moves one triplet along the messenger = TRANSLOCATION. • The movement transfers the deacylated tRNA out of the P site... • moves the peptidyl-tRNA into the P site.

13. Overview • Then the ribosome moves one triplet along the messenger = TRANSLOCATION. • The movement transfers the deacylated tRNA out of the P site... • moves the peptidyl-tRNA into the P site. • Exposes the next codon to be translated in the A site, ready for a new aminoacyl-tRNA to enter.

14. The three stages of translation • Protein synthesis divided into the three stages: • 1.- Initiation • 2.- Elongation • 3.- Termination

15. The three stages of translation Initiation involves the reactions that precede formation of the peptide bond between the first two amino acids of the protein. It requires the ribosome to bind to the mRNA, forming an initiation complex that contains the first aminoacyl-tRNA. This is a relatively slow step in protein synthesis, and usually determines the rate at which an mRNA is translated.

16. The three stages of translation Elongation includes all the reactions from synthesis of the first peptide bond to addition of the last amino acid. Amino acids are added to the chain one at a time. This is the most rapid step in protein synthesis.

17. The three stages of translation Termination encompasses the steps that are needed to release the completed polypeptide chain and dissociate the ribosome from the mRNA.

18. Initiation – Subunit Cycle Ribosomal subunits cycle during prokaryote protein synthesis. Bacterial ribosomes engaged in elongating a polypeptide exist as 70S. At termination, they are released from the mRNA to enter a pool of free ribosomes. In growing bacteria, 80% of ribosomes are synthesizing proteins only 20% are free. Ribosomes in the free pool can dissociate into separate subunits. Free 70S ribosomes are in dynamic equilibrium with 30S and 50S subunits. 20% 80%

19. Initiation - Ribosome recruitment in prokaryotes • The reaction occurs in two steps: • Recognition of mRNA occurs when a small subunit binds to form an initiation complex at the ribosome-binding site. • Then a large subunit joins the complex to generate a complete ribosome.

20. Initiation – Ribosome recruitment in prokaryotes • Initiation prevails when an AUG (or GUG) codon lies within a ribosome-binding site, because only the initiator tRNA can enter the partial P site generated when the 30S subunit binds de novo to the mRNA. • Only the regular aminoacyl-tRNAs can enter the (complete) A site.

21. Initiation – Ribosome recruitment in prokaryotes Initiation involves base pairing between mRNA and rRNA An mRNA contains many AUG triplets: how is the initiation codon recognized as providing the starting point for translation? When ribonuclease is added to the blocked initiation complex, all the regions of mRNA outside the ribosome are degraded. The protected fragments can be recovered and characterized.

22. Initiation – Ribosome recruitment in prokaryotes The protected initiation sequences of bacteria are ~30 bases long. Two common features: The AUG (or less often, GUG or UUG) initiation codon is always included within the protected sequence. Within 10 bases upstream of the AUG is a polypurine stretch known as the Shine-Dalgarno sequence: 5’-AGGAGG-3’ It is complementary to a highly conserved sequence close to the 3’ end of 16S rRNA: 3’-UCCUCC-5’

23. Initiation – Ribosome recruitment in prokaryotes Start Codon AUG Shine-Dalgarno sequence (in mRNA): 5’-AGGAGG-3’ Shine-Dalgarno complementary sequence (16S rRNA): 3’-UCCUCC-5’

24. Initiation – Ribosome recruitment in prokaryotes Mutations of any of these sequences impede ribosome binding. Point mutations in the Shine-Dalgarno sequence prevent mRNA translation. The sequence at the 3’ end of rRNA is conserved between prokaryotes and eukaryotes...

25. Initiation – Ribosome recruitment in prokaryotes However, eukaryotes have a five-base deletion in the complementary rRNA sequence. There is no apparent base pairingbetween eukaryotic mRNA & 18S rRNA. This is a significant difference in the mechanism of initiation.

26. Initiation – Ribosome recruitment In bacteria, a 30S subunit binds directly to the ribosome-binding sites. As most bacterial mRNAs’s are polycistronic, an initiation complex forms at each ORF.

27. Initiation – Shine-Dalgarno Sequences When ribosomes attach to the first coding region, the subsequent coding regions have not yet even been transcribed. By the time the second ribosome site is available, translation is well under way through the first cistron.

28. Initiation – Eukaryotes Virtually all eukaryotic mRNAs are monocistronic.

29. Initiation – Eukaryotes mRNA is usually longer than coding region. The average mRNA is 1000-2000 bases long Methylated cap at the 5’ terminus 100-200 poly-A at the 3’ terminus.

30. Initiation – Eukaryotes The nontranslated 5’ leader is relatively short, usually <100 bases.

31. Initiation – Eukaryotes The nontranslated 3’ trailer is often rather long (100 - 1000 b). By virtue of its location, the leader cannot be ignored during initiation, but the function for the trailer is less obvious.

32. Initiation – Eukaryotes The first feature to be recognized during translation of a eukaryotic mRNA is the methylated cap that marks the 5’ end. Messengers whose caps have been removed are not translatedefficiently. Binding of 40S subunits to mRNA requires several initiation factors, including proteins that recognize the structure of the cap.

33. Initiation – Eukaryotes 5'-modifications occurs to almost allcellular or viralmRNAs and are essentialfor their translation in eukaryotic cytoplasm (not for organelles). The sole exception to this rule is provided by a few viral mRNAs (such as poliovirus) that are not capped; only these exceptional viral mRNAs can be translated without caps. Poliovirus infection inhibits the translation of host mRNAs. This is accomplished by interfering with the cap binding proteins that are needed for initiation of cellular mRNAs, but that are superfluous for the noncapped poliovirus mRNA.

34. Initiation – Eukaryotes (Scanning) "scanning" model supposes that the 40S subunit initially recognizes the 5’ cap and then "migrates" along the mRNA. In many mRNAs the cap and AUG are farther apart, in extreme cases ~1000 bases distant. Yet the presence of the cap is still necessary for a stable complex to be formed at the initiation codon.

35. Initiation – Eukaryotes (Scanning) Scanning from the 5’ end is a linear process.

36. Initiation – Eukaryotes (Scanning) When 40S subunits scan the leader region, they melt secondary structure hairpins with stabilities above -30 kcal. Hairpins of greater stability impede or prevent migration.

37. Initiation – Eukaryotes (Kozak consensus) Migration stops when the 40S subunit encounters the AUG initiation codon. Usually, although not always,the first AUG triplet sequence will be the initiation codon. The AUG triplet by itself is not sufficientto halt migration; it is recognized efficiently as an initiation codon only when it is in the right context. The optimal context consists of the sequence GCCAGCCAUGG “Kozak Consensus Sequence”

38. Initiation – Eukaryotes (Kozak consensus) “Kozak Consensus Sequence” GCCAGCCAUGG The purine (A or G) 3 bases before the AUG codon, and the G immediately following it, are the most important, and influence efficiency of translation by 10X ; the other bases have much smaller effects.

39. Initiation – Eukaryotes (Kozak consensus) When the leader sequence is long, further 40S subunits can recognize the 5’ end before the first has left the initiation site, creating a queueof subunits proceeding along the leader to the initiation site.

40. Initiation – Initiator tRNA • Usually the initiation codon is the triplet AUG, but in bacteria, GUG or UUG are also used. • The AUG codon represents methionine and initiations. • How are they differentiated by translational machinery?

41. Initiation – Initiator tRNA • Two types of tRNA can carry an anticodon for these codons. • One tRNA is used for initiation, the other for recognizing AUG codons during elongation. • Prokaryote initiator tRNA is formylated (thus the • name tRNAf) in aminoacid and tRNA moities. • fMet-tRNAf. • Eukaryote initiator is only formylated in the tRNA.

42. Initiation – Initiator tRNA • What features distinguish the tRNAfMet initiator and the tRNAmMet elongator? • Some of these features are needed to prevent the initiator from being used in elongation, others are necessary for it to function in initiation

43. Initiation – Initiator tRNA The bases C:A on the acceptor stem are NOT paired in tRNAfMet. The absence of this pairing makestRNAfMet uncapable of being incorporated during elongation. Mutations that create a base pair in this position of tRNAfMet allow it to function in elongation. It is also needed for the formylation reaction. Acceptor Stem base-pairing

44. Initiation – Initiator tRNA A series of 3 G:C pairs in the stem that precedes the loop containing the anticodon is unique to tRNAfMet. Are requiredto allow the tRNAfMet to be inserted directly into the P site. Anti-codon stem base-pairing

45. Initiation – Initiator tRNA • In an initiation complex, the small subunit alone is bound to mRNA. • The initiation codon lies within the part of the P site. • The only aminoacyl-tRNA that can become part of the initiation complex is the initiator, which has the unique property of being able to enter directly into the partial P site to recognize its codon.

46. Initiation – Initiator tRNA • This tRNA is used only for initiation. • It recognizes the codons AUG or GUG (occasionally UUG). • The codons are not recognized equally well: the extent of initiation declines 50% when AUG is replaced by GUG. • Inititation declines by 75% when AUG is replaced by UUG. • The species responsible for recognizing AUG codons in internal locations is tRNAmMet. • This tRNA responds only to internal AUG codons. • Its methionine cannot be formylated.

47. Initiation – Initiator tRNA • So there are two differences between the initiating and elongating Met-tRNAs: the tRNA moieties themselves are different; and the amino acids differ in the state of the amino group. • The meaning of the AUG and GUG depends on their context. • When the AUG codon is used for initiation, it is read as formyl-Met; when used within the coding region, it represents Met. • The meaning of the GUG codon is even more dependent on its location. • When present as the first codon, it is read via the initiation reaction as formyl-Met yet when present within a gene, it is read asVal.

48. Initiation events in prokaryotes • The 30S subunit is involved in initiation but not by itself competent to undertake the reactions of binding mRNA and tRNA. • Requires additional proteins called initiation factors (IF). • These factors are found only on 30S subunits, and they are released when the 30S subunits associate with 50S subunits to generate 70S ribosomes.

49. Initiation events in prokaryotes • Initiation in bacteria needs 30S subunits and accessory factors • Initiation factors (IF in prokaryotes, eIF in eukaryotes) are proteins that associate with the small subunit of the ribosomeduring initiation. • Initiation of protein synthesis is not a function of intact ribosomes, but is undertaken by the separate subunits, which reassociate during the initiation reaction. • See “Ribosomal subunit cycle during protein synthesis in bacteria”.

50. Initiation events in prokaryotes • Different sets of accessory factors assist the ribosome at each stage. • Energy is provided at various stages by the hydrolysis of GTP.