2. 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.

3. 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)

4. 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 base4 1 = 4not big enough 2 bases4 2 = 16not big enough 3 bases4 3 = 64THIS WOULD WORK The code could be overlapping or NONOVERLAPPING Nonoverlapping is less sensitive to mutation

5. 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**

6. Reading the codon table

7. 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

8. 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

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

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

11. Charging tRNAs and proofreading tRNA synthetase enzymes can proofread –Can hydrolyze wrong amino acid from tRNA

12. 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

13. Translation mechanism: bacteria INITIATION –Small subunit binds “Shine-Dalgarno” sequence in mRNA 5’-AGGAGG-3’ DNA: 5’-…TATAAT n n n n A n n n n AGGAGG n n n n n ATG…-3’ -10 +1 mRNA: 5’-A n n n n AGGAGG n n n n n AUG…-3’

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

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

16. 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

17. 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 SCANS 5’--> 3’ for the consensus sequence 5’-CCACCAUG-3’ start codon

18. 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

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

21. 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

22. 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) –Peptidyl transferase activity would yield Met-Phe-Ser-tRNA in A site –And so on…

23. Translation mechanism: bacteria TERMINATION –Stop codons are not recognized by any wildtype tRNAs –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

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

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

26. Frameshift mutations Consider the following mRNA sequence Frameshift mutations insert or delete one or more bases into an Open Reading Frame (ORF) –Insert one base –Delete one base 5’-GCCUCAGGAACCACC AUG CUA GCU UGC UGAAAUAAAAAAAAAAA-3’ TLN: M L A C *(stop) 5’-GCCUCAGGAACCACC AUG CUC AGC UUG CUG AAA UAA AAAAAAAAA TLN: M L S L L K *(stop) 5’-GCCUCAGGAACCACC AUG UAG CUUGCUGAAAUAAAAAAAAAAA-3’ TLN: M *(stop)

27. Nonsense mutations & nonsense mediated decay 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 AUG CUA UGG UGC UGAAAUAAAAAAAAAAA-3’ TLN: M L W C *(stop) 5’-GCCUCAGGAACCACC AUG CUA UGA UGC UGAAAUAAAAAAAAAAA-3’ TLN: M L *(stop)

28. 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 X EJC XX