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Definitions

Definitions. tran·scrip·tion (noun): the act of making an exact copy of a document. Example: the very old method for making a copy of a book by hand. trans·la·tion (noun): the rendering of the meaning of something into a different language.

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  1. Definitions • tran·scrip·tion (noun): the act of making an exact copy of a document. • Example: the very old method for making a copy of a book by hand. • trans·la·tion (noun): the rendering of the meaning of something into a different language. • Example: translating Leo Tolstoy’s novel “War and Peace” from Russian (the original) into English.

  2. Translation • The synthesis of a protein polymer from a RNA template • The ribosome translates the chemical language of nucleic acids to amino acids • Provides a control point for regulation of gene expression • Amplification step (can make many protein copies)

  3. Translation • There must be a nucleic acid code for amino acid sequences • 4 different nucleic acid bases, 20 different amino acids • PLUS, need information about where to START and where to STOP translating • Possible CODON sizes: • 1 base 41 = 4 not big enough • 2 bases 42 = 16 not big enough • 3 bases 43 = 64 THIS WOULD WORK • The code could be overlapping or NONOVERLAPPING • Nonoverlapping is less sensitive to mutation

  4. Translation • Codons are nonoverlapping 3 nucleotide units • START = AUG (Methionine) • STOP = UGA, UAG, UAA (does NOT also encode an amino acid) • 61 of 64 codons are left for amino acids • There are only 20 amino acids • The code is “degenerate” with several codons per amino acid • CUN = Leucine • UCN = Serine • CCN = Proline • ACN = Threonine (ACA, ACG, ACC, ACU) (Where N = A, G, C or U) **Note, much of the degeneracy is in the 3rd position of the codon**

  5. Reading the codon table

  6. Transfer RNAs (tRNA) • Bridge between nucleic acid and amino acid languages • 73 - 93 nts long • Several modified bases (e.g. pseudouridine, etc) • Complementary regions base pair to form cloverleaf-like structure • Packs further to look like:  • Amino acid attached to 3’-OH via ester linkage • Anticodon loop basepairs with mRNA codon

  7. Transfer RNAs (tRNA) • Degeneracy of code • Lots of tRNA genes • 1 tRNA can recognize > 1 codon • Strict base pair rules for codon position 1 and 2 • “wobble” in position 3 • Non Watson-Crick pairing e.g. G:U pairing

  8. Transfer RNAs (tRNA) • Examples of tRNAs tolerating G:U pairs in codon position 3

  9. Charging tRNAs • Accuracy for protein synthesis is primarily from the accuracy of attaching the correct amino acid to the correct tRNA • 20 AminoacyltRNAsynthetase enzymes • 1 enzyme for each amino acid • 1 enzyme can recognize >1 tRNA • Specificity from interactions with acceptor and anticodon arms of tRNA

  10. Charging tRNAs and proofreading • tRNAsynthetase enzymes can proofread • Can hydrolyze wrong amino acid from tRNA

  11. The ribosome • Large RNA-Protein complex • Large ribosomal subunit (60S) • Small ribosomal subunit (40S) • Steps in translation: INITIATION • Bind mRNA, find start ELONGATION • Find next amino acid, add it TERMINATION • Recognize stop, and release

  12. Translation mechanism: bacteria • INITIATION • Small subunit binds “Shine-Dalgarno” sequence in mRNA 5’-AGGAGG-3’ DNA: 5’-…TATAAT nnnnAnnnn AGGAGG nnnnn ATG…-3’ -10 +1 mRNA: 5’-A nnnn AGGAGG nnnnn AUG…-3’

  13. Translation mechanism: bacteria • INITIATION • Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG • INITIATION FACTORS 1) IF1, IF2, IF3 • IF2 binds GTP

  14. Translation mechanism: bacteria • INITIATION • Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG • INITIATION FACTORS 1) IF1, IF2, IF3 • IF2 binds GTP 2) IF2 binds initiating tRNA-Met

  15. Translation mechanism: bacteria • INITIATION • Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG • INITIATION FACTORS 1) IF1, IF2, IF3 • IF2 binds GTP 2) IF2 binds initiating tRNA-Met 3) Recruit large subunit, release IF1, IF3 • If codon-anticodon interaction is correct, IF2 hydrolyzes GTP and leaves

  16. Translation mechanism: eukaryotes • INITIATION • A pre-formed eIF1, 2, 3 + Small subunit complex binds 5’-CAP region • eIF4 and other factors are involved in sensing additional features of mRNA • 5’-CAP structure • 3’-end polyA tail • Complex SCANS5’--> 3’ for the consensus sequence 5’-CCACCAUG-3’ start codon

  17. Translation mechanism: bacteria • ELONGATION • After large subunit bound, 3 sites are present in ribosome • Aminoacyl (A) site • Peptidyl (P) site • Exit (E) site • tRNA-MET in P site 1) EF-Tu + GTP + Phe-tRNA bind in A site • If codon-anticodon interaction is proper, hydrolyze GTP --> GDP, release EF-Tu

  18. Translation mechanism: bacteria • ELONGATION • If codon-anticodon interaction is proper, hydrolyze GTP --> GDP, release EF-Tu 2) Peptidyltransferase enzyme catalyzes bond formation • large subunit of Ribosome RNA is a Ribozyme! • Met is now attached at the “A” site: Met-Phe-tRNA

  19. Translation mechanism: bacteria • ELONGATION • Whole ribosome must be translocated 3 nts downstream on mRNA 3) EF-G hydrolyzes GTP for translocation reaction • tRNA in P site is now in E • Met-Phe-tRNA is now in P • Repeat ELONGATION cycle until a stop codon is reached

  20. Translation mechanism: bacteria • ELONGATION • Repeat ELONGATION cycle until a stop codon is reached • EF-Tu + GTP + Ser-tRNA --> EF-Tu + GDP (note exit of tRNA from E site) • Peptidyltransferase activity would yield Met-Phe-Ser-tRNA in A site • And so on…

  21. Translation mechanism: bacteria • TERMINATION • Stop codons are not recognized by any wildtypetRNAs • Three Release Factor proteins • RF1 • RF2 • RF3 • Enter A site and trigger hydrolysis of Met-Phe-Ser from tRNA • Large and small subunits dissociate from mRNA template U A A

  22. Polyribosomes • mRNAs can be translated by multiple ribosomes at same time • Amplification step in gene expression

  23. Coupled TXN & TLN: bacteria • A gene can be transcribed and translation of it can start before TXN is finished

  24. Frameshift mutations 5’-GCCUCAGGAACCACC AUGCUAGCUUGCUGAAAUAAAAAAAAAAA-3’ TLN: M L A C *(stop) • Consider the following mRNA sequence • Frameshiftmutations insert or delete one or more bases into an Open Reading Frame (ORF) • Insert one base • Delete one base 5’-GCCUCAGGAACCACC AUGCUCAGCUUGCUGAAA UAA AAAAAAAAA TLN: M L S L L K *(stop) 5’-GCCUCAGGAACCACC AUGUAGCUUGCUGAAAUAAAAAAAAAAA-3’ TLN: M *(stop)

  25. Nonsense mutations & nonsense mediated decay 5’-GCCUCAGGAACCACC AUGCUAUGGUGCUGAAAUAAAAAAAAAAA-3’ TLN: M L W C *(stop) • Consider the following mRNA sequence • Nonsense mutations change an amino acid codon to a stop codon • If this mutation is in any exon other than the last one, • Nonsense mediated decay (NMD) will block translation of it 5’-GCCUCAGGAACCACC AUGCUAUGAUGCUGAAAUAAAAAAAAAAA-3’ TLN: M L *(stop)

  26. Nonsense mutations & nonsense mediated decay • Exon-Junction Complex (EJC) proteins are deposited on transcripts ~20 nts upstream of new Exon-Exon junctions • Ribosome knocks them off during TLN • If ribosome doesn’t knock them off, transcript is destroyed • Wildtype mRNA: • Nonsense mutation mRNA: • EJC that is not removed generates a signal targeting mRNA for destruction EJC EJC EJC X EJC EJC EJC X X

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