1. 7. Protein Synthesis and the Genetic Code a). Overview of translation i). Requirements for protein synthesis ii). messenger RNA iii). Ribosomes and polysomes iv). Polarity of protein synthesis b). Transfer RNA i). tRNA as an adaptor ii). Amino acid activation iii). Aminoacyl tRNA synthetases iv). “Charged” tRNA c). The genetic code i). Codon-anticodon interactions ii). Initiation codon in prokaryotes vs. eukaryotes iii). Reading frame d). Mutations affecting translation i). Frameshift mutations ii). Missense and nonsense mutations

2. Overview of translation last step in the flow of genetic information definition of translation requirements for protein synthesis mRNA ribosomes initiation factors elongation and termination factors GTP aminoacyl tRNAs amino acids aminoacyl tRNA synthetases ATP

3. Messenger RNA (mRNA) m 7 Gppp Cap 5’ 5’ untranslated region AUG initiation codon translated (coding) region (AAAA) n poly(A) tail 3’ untranslated region UGA termination codon 3’AAUAAA

4. Ribosomes prokaryotic ribosome eukaryotic ribosome 70S ribosome 80S ribosome 50S subunit 23S rRNA 5S rRNA 35 proteins 60S subunit 28S rRNA 5S rRNA 5.8S rRNA 49 proteins 30S subunit 16S rRNA 21 proteins 40S subunit 18S rRNA 33 proteins

5. Polysomes direction of translation is 5’ to 3’ along the mRNA direction of protein synthesis is N terminus to C terminus UGA5’ large ribosomal subunit small ribosomal subunit AUG polysome nascent polypeptide N N subunits dissociate

6. Transfer RNA tRNA is the “adaptor” molecule in protein synthesis acceptor stem CCA-3’ terminus to which amino acid is coupled carries amino acid on terminal adenosine anticodon stem and anticodon loop

7. Amino acid activation and aminoacyl tRNA synthetases aminoacyl tRNA synthetases are the enzymes that “charge” the tRNAs 20 amino acids one aminoacyl tRNA synthetase for each amino acid can be several different “isoacceptor” tRNAs for each amino acid all isoacceptor tRNAs for an amino acid use the same synthetase each aminoacyl tRNA synthetase binds amino acid ATP isoacceptor tRNAs

8. H 2 N-C-C-OH H R - - O = ATP H 2 N-C-C-O-P-O-ribose-adenine H R - - O = amino acid adenylated (activated) amino acid PPi uncharged tRNA H 2 N-C-C-O H R - - O = aminoacyl (charged) tRNA AMP 3’ Amino acid activation and tRNA charging

9. The genetic code consists of 64 triplet codons (A, G, C, U) 4 3 = 64 all codons are used in protein synthesis 20 amino acids 3 termination (stop) codons: UAA, UAG, UGA AUG (methionine) is the start codon (also used internally) multiple codons for a single amino acid = degeneracy 5 amino acids are specified by the first two nucleotides only

10. The Genetic Code UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Phe Leu Val Ile Met Ser Pro Thr Ala Tyr Stop His Gln Asn Lys Asp Glu Cys Arg Ser Arg Gly Stop Trp

11. Codon-anticodon interactions codon-anticodon base-pairing is antiparallel the third position in the codon is frequently degenerate one tRNA can interact with more than one codon (therefore 50 tRNAs) wobble rules C with G or I (inosine) A with U or I G with C or U U with A, G, or I I with C, U, or A 5’3’ A U G U A C 3’5’tRNA met mRNA 5’3’ C U A G G A U 3’5’tRNA leu mRNA wobble base one tRNA leu can read two of the leucine codons

12. Inosine = Cytidine Inosine = Adenosine Inosine = Uridine Guanosine = Uridine Wobble Interactions

13. Initiation in prokaryotes and eukaryotes initiation can occur at internal AUG codons in prokaryotic mRNA initiation in eukaryotes occurs only at the first AUG codon lac operon in E. coli is transcribed as a polycistronic mRNA with multiple AUG codons eukaryotic mRNA lac IPOlac Zlac Ylac A AUG SDAUGSDAUG initiation codon with Shine-Dalgarno site initiation codon with Shine-Dalgarno site internal Met codon does not have Shine-Dalgarno site 5’ 5’ cap AUG initiation can only occur at first AUG codon downstream of the 5’ cap AUG internal (downstream) Met codon cannot serve as an initiation site AUG

14. Reading frame reading frame is determined by the AUG initiation codon every subsequent triplet is read as a codon until reaching a stop codon...AGAGCGGA.AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA... MET.ALA.GLU.TRP.LEU.SER.MET.SER a frameshift mutation...AGAGCGGA.AUG.GCA.GA.UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA... the new reading frame results in the wrong amino acid sequence and the formation of a truncated protein...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA... MET.ALA.ASP.GLY

15. Mutations affecting translation hemoglobin Wayne (3’ terminal frameshift mutation) Normal  -globin.ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA GCC UCG GUA.THR.SER.LYS.TYR.ARG Wayne  -globin.ACG.UCA.AAU.ACC.GUU.AAG.CUG.GAG.CCU.CGG.UAG.THR.SER.ASN.THR.VAL.LYS.LEU.GLU.PRO.ARG mutated region missense mutations (e.g., AGC Ser to AGA Arg) nonsense mutations (e.g., UGG Trp to UGA Stop) read through, reverse terminator, or sense mutations (e.g., UAA Stop to CAA Gln) as in hemoglobin Constant Spring silent mutations (e.g., CUA Leu to CUG Leu) do not affect translation