1. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 TRANSLATION

2. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Protein synthesis in bacteria Dr Mike Dyall-Smith, lab 3.07, mlds@unimelb.edu.au Aims: Understand the process of translation in Bacteria Know the the major components involved Know the typical genetic signals needed to allow translation of a mRNA Familiar with the process of protein folding http://mmbr.asm.org/cgi/content/full/69/1/101#Initiator_tRNA References:, Schaecter et al., Microbe, pp149-58 or Any recent molecular biology textbook. A recent review is: http://mmbr.asm.org/cgi/content/full/69/1/101#Initiator_tRNA

3. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Lecture outline: 1. Overview of translation 1. Overview of translation in bacteria (comparison with eucarya) components, genetic code, process. 2. Features of mRNA and tRNA 4. Ribosome structure and function 4. Ribosome structure and function (compared to eucarya) 5. Initiation and termination factors 5. Initiation and termination factors and the process of translation 6. Briefly touch on protein folding. *export and processing covered in future lecture.

4. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Central dogma Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 DNA RNA PROTEIN Transcription Translation

5. Translation The decoding (translation) of the 3-letter code of nucleotide bases in the mRNA, to direct the synthesis of proteins. A much more complex operation than transcription.

6. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Overall process of translation in bacteria Initiation Elongation Termination Initiation factors mRNA Requires aa-tRNAs, ATP, GTP Speed: ~ 15 aa/sec

7. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Protein synthesis in bacteria How do ribosomes bind to mRNA? Small subunit binds initiation factorsSmall subunit binds initiation factors This complex binds to mRNA, initiator tRNA can then bind.This complex binds to mRNA, initiator tRNA can then bind. The large subunit joins the complexThe large subunit joins the complex Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

8. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Transfer RNA Acceptor Arm Anticodon Extra Arm D Arm T  Arm Amino acid added at 3’ end

9. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Transfer RNA (tRNA) structure Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Linkage to aa tRNA Arm Anticodon

10. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 tRNA is actually ‘L’ shaped Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Anticodon arm

11. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Picture from Schaecter et al. For use by students enrolled in 526-301 UNIVERSAL GENETIC CODE

12. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Ribosomes are large enough to bind 2 tRNAs and cover 40 nt of mRNA

13. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

14. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

15. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 70 S Ribosomes

16. Ribosomes contain many proteins and 3 rRNAs 2/3 of the mass of bacterial ribosomes is rRNA

18. Initiation of translation:

21. IF-2 puts the fMet-tRNA f initiator in the P site. IF-2, bound to GTP, associates with the P site of the 30S subunit. fMet-tRNA f. then binds to the IF- 2 on the 30S subunit. fMet-tRNA f. then binds to the IF- 2 on the 30S subunit. IF-2 then transfers the tRNA into the partial P site 50S subunit binds and IF1-3 are released.

22. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Where do ribosomes bind on mRNA ? Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

23. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Ribosome binding sites bind to the 3’ end of 16S rRNA Shine-Dalgarno sequence Also called ribosome binding site or RBS

24. Initiator tRNA in bacteria is tRNA f Initiator tRNA in bacteria is tRNA f MET N-formyl-methionyl tRNA. It is only used for initiating translation. All proteins start with this amino acid. Internal methionines use a different tRNA, tRNA m MET

25. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 tRNA and mRNA move in the same direction through the ribosome Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

26. A site : site where an aminoacyl-tRNA enters to base pair with a codon. P site : site occupied by a peptidyl-tRNA Deacylated tRNA: has no amino acid or polypeptide chain attached Translocation: the movement of the ribosome, one codon at a time, along mRNA after the addition of an amino acid to the polypeptide chain. Elongation: the stage in a macromolecular synthesis reaction (replication, transcription, or translation) when the nucleotide or polypeptide chain is extended by the addition of individual subunits.

27. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Two sites for binding tRNAs on the ribosome P A Peptide bond formed Translocation P and A sites occupied Ribosome movement

28. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Typical polycistronic bacterial mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

29. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Reinitiation on polycistronic mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

30. Termination at stop codons: UAA, UGA, UAG Termination codons are recognised by ‘release factor’ proteins (RF1/RF2, RF3). RF1 or RF2 bind to stop codons in the A site of ribosomes, and activate the ribosome to hydrolyse the adjacent peptidyl tRNA (in the P site), to release the protein. RF3 releases the RF1 or RF2. Finally, RRF (ribosome recycling factor) dissociates the remaining mRNA, tRNA and ribosome subunits.

32. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 mRNA degradation in bacteria is rapid Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Half-life of 1-3 min

33. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 mRNA degradation in bacteria is rapid Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Q: What happens if the ribosome reaches the end of a broken mRNA without seeing a stop codon ? A: it gets stalled !! TGA

34. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 tmRNA - how to overcome stalled ribosomes Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 tmRNA is part tRNA part mRNA. It completes translation, putting a small peptide sequence at the end of a protein. This is a tag for protein degradation. http://www.indiana.edu/~tmrna/

35. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Overall process of translation in Bacteria Initiation Elongation Termination Initiation factors mRNA Requires aa-tRNAs, ATP, GTP Speed: ~ 15 aa/sec

36. Protein folding Most proteins will not fold correctly without assistanceMost proteins will not fold correctly without assistance Cell has several chaperones that perform that functionCell has several chaperones that perform that function Otherwise, misfolded proteins are degraded by proteasesOtherwise, misfolded proteins are degraded by proteases

37. Protein folding Trigger factor: peptidyl proline isomeraseTrigger factor: peptidyl proline isomerase DnaK: removes small, misfolded hydrophobic regionsDnaK: removes small, misfolded hydrophobic regions GroEL and GroES: major folding/refolding, protected from proteasesGroEL and GroES: major folding/refolding, protected from proteases

38. Protein folding

39. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Lecture outline: 1. Overview of translation 1. Overview of translation in bacteria (and comparison with eucarya) components, genetic code, process. 2. Features of mRNA and tRNA 3. Ribosome structure and function 3. Ribosome structure and function (compared to eucarya) 4. Initiation and termination factors 4. Initiation and termination factors and the process of translation 5. Outline of protein folding Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301