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By the end of this series of slides, you should be able to explain much of this animation http://www.crocoduck.bch.mssta

Translation. By the end of this series of slides, you should be able to explain much of this animation http://www.crocoduck.bch.msstate.edu/EMG/Translation_588x392.swf. The Genetic Code. Degeneracy and synonyms Minimizing impacts of mutation Similar codon – silent mutation or

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By the end of this series of slides, you should be able to explain much of this animation http://www.crocoduck.bch.mssta

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  1. Translation By the end of this series of slides, you should be able to explain much of this animationhttp://www.crocoduck.bch.msstate.edu/EMG/Translation_588x392.swf

  2. The Genetic Code • Degeneracy and synonyms • Minimizing impacts of mutation • Similar codon – • silent mutation or • similar amino acid Acidic Basic Neutral-polar (+Cys) Neutral-nonpolar (+Pro, Gly)

  3. Translation • Codon – Anticodon pairing • The first two positions must pair exactly but the third is more relaxed • Anticodon U can pair with A or G on mRNA • Anticodon I (derived from G) can pair with U, C, or A • Allows for fewer required tRNAs • Leucine (6 codons) requires only 3 different tRNAs

  4. The Genetic Code • Cracking the code • Early 1960s • Synthetic polynucleotide RNAs + cell extract  amino acid chains • Single polynucleotide chains  single amino acid polymers • Poly-U – phe • Poly-A – lys • Poly-G – Gly • Poly-C – Pro • No method to order specific codons at the time

  5. The Genetic Code • Cracking the code • Early 1960s • Mixed copolymers • Vary ratios of two nucleotides to generate mixed polynucleotide chains • 2:1 ratio of A:C  lots of CAC, CCA, ACC codons •  histidine/threonine prevelance • Suggests potential codons for histidine • More precision needed

  6. The Genetic Code • Cracking the code • Mid 1960s, defined codons • Nirenberg and Leder (1964) bound specific individual triplets to ribosomes • Ribosomes in turn bound individual amino acids • Some triplets not efficient binders

  7. The Genetic Code • Cracking the code • Mid 1960s, defined codons • Repeating copolymers

  8. The Genetic Code • Three rules • 1. Codons are read in a 5’-3’ direction • 2. Codons are non-overlapping with no gaps • 3. There is a fixed reading frame relative to the start codon

  9. The Genetic Code • Point mutations • Missense mutations – changing one amino acid to another • Nonsense (stop) mutations – change an amino acid to a stop • Frameshift mutations – alter the reading frame • Suppressor mutations reinstate the correct amino acid chain (at least partially) • Back mutations • Intragenic mutations – compensatory mutations • Intergenic mutations – involves mutant tRNAs

  10. The Genetic Code • Nearly universal • Very well conserved but some subcellular organelles show variation

  11. Translation • Getting the information contained in an mRNA converted into a protein • In bacteria, up to 80% of energy devoted to translation • Machinery • mRNA • tRNA • Aminoacyl-tRNAsynthetase • ribosomes

  12. Translation • Anatomy of an mRNA • Open Reading Frames, ORFs • Bounded by start • 5’-AUG-3’ in eukaryotes • and stop codons • UAG, UGA, UAA • Polycistronic vs. monocistronic • UTRs • Introns and exons

  13. Translation • Anatomy of an mRNA • Prokaryotes • Ribosome binding site (RBS) • 3-9 bp upstream of start codon • Complementary sequence on 16S rRNA • Alterations strengthen or weaken the RBS

  14. Translation • Anatomy of an mRNA • Eukaryotes • Ribosomes recruited via 5’ cap • Scanning • Poly-A tail enhances translation

  15. Translation • tRNAs • Adapters b/t mRNA codons and amino acids • 75-95 nt long • 3’ terminus = 5’-CCA-3’ • Amino acid attachment • Sometimes added post-transcriptionally via CCA adding enzyme • Common shared secondary structure

  16. Translation • tRNAs • Modified bases created post-transcriptionally • Pseudouridine (ΨU) • Dihydrouridine (D) • Hypoxanthine, inosine, methylguanosine, thymine

  17. Translation • tRNAs • ΨU-loop • D-loop • Acceptor arm • Anticodon loop • Variable loop – 3-21 nt • 3D structure is L-shaped

  18. Translation • tRNA charging • Carboxyl group to 2’ or 3’ OH of A from tRNA • Aminoacyl-tRNAsynthetase

  19. Translation • tRNA charging • Synthetases must • Recognize correct tRNA • Recognize correct amino acid • tRNA recognition • No common set of rules across all tRNAs • One or more discriminator base(s) • One or more anticodon base(s) • One or more bases in acceptor stem • Various other “inside the L” bases Important recognition sites for some tRNAs

  20. Translation • tRNA charging • Synthetases must • Recognize correct tRNA • Recognize correct amino acid • Amino acid recognition • Coarse recognition via size/chemical groups

  21. Translation • tRNA charging • Synthetases must • Recognize correct tRNA • Recognize correct amino acid • Amino acid recognition • Small aa’s can fit into large pockets and lead to mischarging • Editing • Second recognition pocket can accommodate small aa’s but not large aa’s • Hydrolyze (cut off) aa’s that fit into pocket • Process repeated until aa added that can’t fit in pocket

  22. Translation • tRNA charging • Ribosomes cannot identify mischarged tRNAs • 1962 experiment • tRNA-ACA normally charged with Cys • Develop cell-free extract with Cys-tRNA-ACA • Introduce metal catalyst to change charged Cys to Ala • Ribosomes cannot recognize mischarged tRNAs RNA template UGUGUGUGUG... polyCys chain RNA template UGUGUGUGUG... polyAla chain

  23. Translation • Ribosomes • Massive – three+ RNAs and 50 proteins • Large and small subunits • Large - Peptidyltransferase center • Small – decoding center • Measured in Svedberg units (S) corresponding to sedimentation velocity • Prokaryotic • Total - 70S • Small – 30S • Large – 50S • Eukaryotic • Total - 80S • Small - 40S • Large – 60S • Part II of structural tutorial

  24. Translation • Ribosomes • RNA components also measured using S

  25. Translation • Ribosomes • The ribosome cycle

  26. Translation • Polyribosomes Prokaryote Eukaryote Note the difference – Due to presence/absence of nuclear membrane

  27. Translation • Ribosomes • Three tRNA binding sites • A-site – aminoacyl-tRNA • P-site –peptidyl-tRNA • E-site – exit • 3’ termini of A and P tRNAs very close • Structural tutorial part III

  28. Translation • Ribosomes • Codon-anticodon interactions in the small subunit • Structural tutorial part III

  29. Translation • Ribosomes • Channels through ribosome allow mRNA entry/exit, tRNA entry/exit, and polypeptide exit

  30. Translation • Translation initiation • Three events: • Ribosome recruitment • Start codon positioning • tRNA brought to P site • Different processes in prokaryotes and eukaryotes

  31. Translation • Translation initiation • Prokaryotes • Base pairing positions the small subunit

  32. Translation • Translation initiation • Prokaryotes • Initiation factors • IF1 – blocks tRNAs from binding to A-site • IF2 – escorts initiator tRNA, GTPase • IF3 - blocks large subunit from small subunit

  33. Translation • Translation initiation • Eukaryotes • Four steps: • Binding of initiator tRNA • Auxiliary factors bind to mRNA • Bound ribosome scans for start codon (1st AUG) • Large subunit recruited • 1. eIF1, eIF1A, eIF3, & eIF5 associate with small subunit • Analogous to IF1 & IF3 • 2. eIF2 escorts initiator tRNA

  34. Translation • Translation initiation • Eukaryotes • Four steps: • Binding of initiator tRNA • Auxiliary factors bind to mRNA • Cap recognized by eIF4E • eIF4A, eIF4G • Helicase activity of eIF4A activated by eIF4B • Bound ribosome scans for start codon (1st AUG) • Large subunit recruited

  35. Translation • Translation initiation • Eukaryotes • Four steps: • Binding of initiator tRNA • Auxiliary factors bind to mRNA • Bound ribosome scans for start codon (1st AUG) • Large subunit recruited

  36. Translation • Translation initiation • Eukaryotes • Four steps: • Binding of initiator tRNA • Auxiliary factors bind to mRNA • Bound ribosome scans for start codon (1st AUG) • Large subunit recruited • 1. ATP dependent movement of small subunit toward start codon • 2. Codon recognized by anticodon • 3. Release of eIF1, eIF2, eIF4B, eIF5 mediated by hydrolysis of eIF2-GTP • 4. eIF5B-GTP recruited by eIF1A

  37. Translation • Translation initiation • Eukaryotes • Four steps: • Binding of initiator tRNA • Auxiliary factors bind to mRNA • Bound ribosome scans for start codon (1st AUG) • Large subunit recruited • 1. eIF5B recruits large subunit • 2. GTP hydrolysis releases eIF1A and eIF5B

  38. Translation • Translation initiation • Eukaryotes • mRNA is circularlized via a protein bridge b/t eIF4G and PABP

  39. Translation • Translation elongation • Prokaryotes • Three steps: • tRNA binding at A site • Peptide bond formation • translocation

  40. Translation • Translation elongation • Prokaryotes • Three steps: • tRNA binding at A site • Peptide bond formation • translocation • EF-Tu – escorts tRNAs to A-site • GTP hydrolysis reduces affinity of EF-Tu for tRNA • Hydrolysis via GTPase domains on ribosome and EF-Tu

  41. Translation • Translation elongation • Prokaryotes • Three steps: • tRNA binding at A site • Peptide bond formation • translocation • EF-Tu – escorts tRNAs to A-site • GTP hydrolysis reduces affinity of EF-Tu for tRNA • Hydrolysis via GTPase domains on ribosome and EF-Tu • Incorrect codon/anticodon match does not position GTPase domain correctly

  42. Translation • Translation elongation • Prokaryotes • Three steps: • tRNA binding at A site • Peptide bond formation • translocation • Accommodation – rotation of the acceptor end of tRNA to bring aa into proximity with peptide chain • Some amino acid participation in peptide bond formation but primarily RNA driven

  43. Translation • Ribosomes • The peptidyltransferase reaction • During aa chain growth, two charged amino acids are housed in the ribosome • Peptidyl-tRNA – carries the aa just added to the chain; still attached to the tRNA • Aminoacyl-tRNA – the next one to be added • Peptidyltransferase reaction breaks tRNA-aa bond on peptidyl-tRNA • RNA is the catalyst

  44. Translation • Translation elongation • Prokaryotes • Three steps: • tRNA binding at A site • Peptide bond formation • translocation • Translocation driven by EF-G, peptidyltransferase reaction, and GTP hydrolysis • Peptidyltransferase shifts acceptor end into P site but not anticodon end • EF-G enters empty factor binding site • Hydrolysis changes EF-G conformation to push tRNA out of A site EF-Tu + tRNA EF-G

  45. Translation • Translation elongation • Eukaryotes • Three steps: • tRNA binding at A site • Peptide bond formation • translocation • Analogous processes and players

  46. Translation • Translation termination • Release factors (RF) • Class I – recognize stop codons and trigger release of polypeptide • RF1 – UAG, UAA; RF2 – UGA, UAA • eRF1 • Peptide anticodon • GGQ (gly,gly,glu) motif likely extends into peptidyltransferase center tRNA, RF1

  47. Translation • Translation termination • Release factors (RF) • Class II – stimulate dissociation of class I factors • RF3, eRF3 • GTP binding and hydrolysis

  48. Translation • Translation termination • Ribosome recycling • RRF – ribosome recycling factor • Recruits EF-G-GTP • Ratchets ribosome apart • All illustrated via animation on website

  49. Translation • Translation difficulties • Nonsense mediated decay • Nonsense codon = early stop codon • Normal mRNA • exon junction complexes displaced by ribosome • Nonsense mRNA • Exon junction proteins not displaced • Recruit Upf proteins to remove 5’ cap • Endonuclease degrades mRNA

  50. Translation • Translation difficulties • Broken mRNAs lead to stalled ribosomes • Stop codon necessary for ribosome release • tmRNA – part tRNA, part mRNA • ssrA RNA – 457 nt • Includes tRNA-like region followed by 10 codons and stop codon • Resulting ‘tagged’ protein is degraded

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