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Translating the Genetic Code Gene expression part 3 An overview of gene expression Figure 13.2 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? The Coding Problem • 1-1 correspondence – NO – 4 possibilities • 2-1 correspondence – NO – 16 possibilities • 3-1 correspondence – YES – 64 possibilities 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 1st 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 Crick’s Experiments to Determine Triplet Nature of Code So.. The code is 3 letter words, but what about punctuation? a b c 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 Any frame is potentially the reading frame! a b c ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW The actual reading frame is called the Open Reading Frame or ORF Reading Frames & Mutation Types • Frame shift mutations – Original reading frame is frame a – Insertions or deletions shift the reading frame a b c ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW ^ a b c ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW Reading Frames & Mutations a b c ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW • 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. ^ a b c ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE W.. 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 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 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 Decoding the Genetic Code • 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 … (%) Amino Acid Incorporated Possible Codons Percentage in the Random Polymer GGG 0.7 x 0.7 x 0.7 = 0.34 = 34% (GGG + GGU, UGG, GUG) GGU 0.7 x 0.7 x 0.3 = 0.15 = 15% Valine GUU 0.7 x 0.3 x 0.3 = 0.06 = 6% (GGU, UGG, GUG, GUU, UUG, UGU) 21 (15 + 6) UUU 0.3 x 0.3 x 0.3 = 0.03 = 3% Tryptophan 15 UGG 0.3 x 0.7 x 0.7 = 0.15 = 15% UUG 0.3 x 0.3 x 0.7 = 0.06 = 6% (UUG, GUU, UGU) UGU 0.3 x 0.7 x 0.3 = 0.06 = 6% Leucine GUG 0.7 x 0.3 x 0.7 = 0.15 = 15% Radiolabeled Amino Acid Added Glycine 49 (34 + 15) (UGG, GUG, GGU) Cysteine 6 6 (UUG, GUU, UGU) Phenylalanine (UUU) 3 Polymers of Nucleotide Repeats Trinucleotide-tRNA Binding Analysis • 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