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TRANSLATION

TRANSLATION. 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

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TRANSLATION

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  1. TRANSLATION Dr Mike Dyall-Smith, 2007

  2. 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 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 Dr Mike Dyall-Smith, 2007

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

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

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

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

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

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

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

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

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

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

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

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

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

  17. Initiation of translation:

  18. Initiation of translation: IF-2 puts the fMet-tRNAf initiator in the P site. IF-2, bound to GTP, associates with the P site of the 30S subunit. fMet-tRNAf. 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.

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

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

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

  22. 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 Dr Mike Dyall-Smith, 2007

  23. 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.

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

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

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

  27. 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.

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

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

  30. tmRNA - how to overcome stalled ribosomes 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/ Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007

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

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

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

  34. Protein folding

  35. Lecture outline: 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 (compared to eucarya) 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 Dr Mike Dyall-Smith, 2007

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