1. Translating the Genetic Code Gene expression part 3

2. Figure 13.2 An overview of gene expression

3. The Idea of A Code 20 amino acids 4 nucleotides How do nucleic acids composed of 4 nucleotides specify the synthesis of proteins composed of 20 different amino acids?

4. The Coding Problem 1-1 correspondence – NO – 4 possibilities 2-1 correspondence – NO – 16 possibilities 3-1 correspondence – YES – 64 possibilities

5. Determination of Triplet Nature of Code Aacridines & flavinoids cause single nucleotide deletions and insertions respectively Insertion of 1 base shifts frame & creates non-sense mutation Deletion of base in mutant restores frame (suppresses 1 st mutation) Recombination between mutants showed that insertion of 1 or 2 bases or removal of 1 or 2 bases doesn't restore frame, but insertion of 3 or removal of 3 allows frame to continue

6. Crick’s Experiments to Determine Triplet Nature of Code

7. So.. The code is 3 letter words, but what about punctuation? GROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW There is a message, but one must start at the right place to read it Code written in three letter words - codon There are three reading frames, but only one gives an intelligible message – frame b –NOW THE CAT SAW THE DOG BUT DID NOT RUN A start codon (NOW) and a stop codon (END) define the frame to use abcabc

8. Any frame is potentially the reading frame! OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW abcabc ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW abcabc The actual reading frame is called the Open Reading Frame or ORF

9. Reading Frames & Mutation Types Frame shift mutations –Original reading frame is frame a –Insertions or deletions shift the reading frame ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW abcabc ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW abcabc ^

10. Reading Frames & Mutations Once a ribosome begins translation in a particular frame (a) it does not shift frames Therefore, if a mutation shifts the reading frame in the mRNA, the ribosome will read the wrong frame. ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW abcabc ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW abcabc ^ NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE W..

11. Deciphering the Code Each amino acid in a protein is specified by 3 nucleotides of codon Each codon specifies only ONE amimo acid There are 64 possible codons but only 20 amino acids Degeneracy –An amino acid can be specified by multiple codons –A given codon still only specifies only one amino acid

12. Deciphering the Code: Three Approaches Synthesis and translation of homopolymer RNA & ratio polymer RNA followed by amino acid analysis Synthesis and translation of di, tri and tetra nucleotide repetitive RNA polymers followed by amino acid analysis Triplet RNA-tRNA binding assay and amino acid analysis

13. Synthetic RNA Templates and In Vitro Translation Synthesis of homopolymeric RNAs –UUUUUUUUUU, AAAAAAAAAA, CCCCCCCCC, & GGGGGGGGG When translated produced polypeptides –poly phenylalanine (UUU), polyproline (CCC), polylysine (AAA), and polyglycine (GGG) Therefore 4 codons were determined –UUU = phe –CCC = pro –AAA = lys –GGG = gly

14. The enzyme polynucleotide phosphorylase –polymerizes ribonucleoside diphosphates (NDPs) into RNA –It does not use a template, the order of nucleotides is random –The nucleotide sequence is controlled by the relative abundance of NDPs For example, if 70% GDP and 30% UDP are mixed together, then … Possible Codons Percentage in the Random Polymer GGG0.7 x 0.7 x 0.7 = 0.34 = 34% GGU0.7 x 0.7 x 0.3 = 0.15 = 15% GUU0.7 x 0.3 x 0.3 = 0.06 = 6% UUU0.3 x 0.3 x 0.3 = 0.03 = 3% UGG0.3 x 0.7 x 0.7 = 0.15 = 15% UUG0.3 x 0.3 x 0.7 = 0.06 = 6% UGU0.3 x 0.7 x 0.3 = 0.06 = 6% GUG0.7 x 0.3 x 0.7 = 0.15 = 15% Decoding the Genetic Code Radiolabeled Amino Acid Added (%) Amino Acid Incorporated Glycine (GGG + GGU, UGG, GUG) 49 (34 + 15) Valine (GGU, UGG, GUG, GUU, UUG, UGU) 21 (15 + 6) Tryptophan (UGG, GUG, GGU) 15 Cysteine (UUG, GUU, UGU) 6 Leucine (UUG, GUU, UGU) 6 Phenylalanine (UUU) 3

15. Polymers of Nucleotide Repeats

16. Trinucleotide-tRNA Binding Analysis

18. Special codons: –AUG (which specifies methionine) = start codon AUG specifies internal methionines also –UAA, UAG and UGA = termination, or stop, codons The code is degenerate –More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for lysine –In most instances, the third base is the degenerate base It is sometime referred to as the wobble base The code is nearly universal –Only a few rare exceptions have been noted