Genetic CodePathway for Gene Expression3 nucleotides encode a single amino acidExperiments to decipher the codeTools availableHomopolymers of RNA direct synthesis of homopolypeptidesResults of using homopolymers to program translationMixed co-polymers of RNA direct the incorporation of particular amino acids into polypeptidesFrequency of incorporation of each amino acid correlates with frequency of occurrence of a particular trinucleotideDefined trinucleotides stimulate binding of particular aminoacyl-tRNAs to ribosomesFeatures of the Genetic CodeTable 3.4.4 The Genetic CodeDegeneracy of the codeGroupings of codonsCodons for initiation of translationCodons for termination of translationGenetic code is univeral (almost)Differential codon usageWobble in anticodon-codon pairingTypes of mutations in coding regionsChanges that alter the encoded productQuizGenetic CodePathway for Gene ExpressionDNApre-mRNAproteintranscriptiontranslationreplicationreverse transcriptionmRNAForm endsSplicepost-translationalmodificationsfunctional protein3 nucleotides encode a single amino acid•For 4 nucleotides to encode 20 amino acids, you need a coding unit of at least 3:–A coding unit of 2 nucleotides can only encode 16 amino acids (4x4)–A coding unit of 3 nucleotides can only encode 64 amino acids (4x4x4)•Insertions or deletions of 1, 2, 4, 5, etc nucleotides cause a severe loss of function resulting from a change in the reading frame.•But insertions or deletions of 3, 6, 9, etc have little effect on the phenotype, because the reading frame is not affected for most of the mRNA.Experiments to decipher the codeTools available•Cell-free systems for translation–From bacteria, plants and animals•Ability to synthesize polyribonucleotides–Polynucleotide phosphorylase can make RNAs from NDPs–Physiological function: reverse reaction for RNA degradationnNDP(NMP)nnPi+Homopolymers of RNA direct synthesis of homopolypeptides•Use of a single NDP as a substrate for polynucleotide phosphorylase will produce a homopolymer of that nucletide–UDP as a substrate results in polyU as the product•Addition of polyU to a cell-free translation system results in polyphenylalanine as the product.Results of using homopolymers to program translation•UUU encodes Phe•AAA encodes Lys•CCC encodes Pro•GGG encodes GlyMixed co-polymers of RNA direct the incorporation of particular amino acids into polypeptides•Mix two NDPs in a known ratio, and polynucleotide phosphorylase will catalyze synthesis of an RNA with those 2 nucleotides in that ratio, but random order.nADP(AMP)n-(CMP)m(n+m)Pi++ mCDPe.g. ADP:CDP 5:1(AMP)5-(CMP)1e.g. AACAAAAACAACAAAAAAAAACAAFrequency of incorporation of each amino acid correlates with frequency of occurrence of a particular trinucleotide•For a 5:1 ratio of ADP:CDP -> poly(AC) (5:1), A is present in the RNA 5/6 of the time, and C is present at a frequency of 1/6.•Possible trinucleotides (codons) and resulting amino acids incorporated during translation:Compo- Num- Relative Amino Freq. sition ber Frequency Freq. Acid of incorp.3A 1 0.579 1.0 Lys 1.02A, 1C 3 3x0.116 3x0.20 Thr*, Asn, Gln 0.27*, 0.24, 0.241A, 2C 3 3x0.023 3x0.04 Pro**, His, Thr* 0.07,0.07, (.04*)3C 1 0.005 0.01 Pro** (0.01**)Defined trinucleotides stimulate binding of particular aminoacyl-tRNAs to ribosomes+*AA-tRNA+ribosomeNNN*AA-tRNANNNBinds to filterWhich trinucleotide will allow binding of a particular AA-tRNA to ribosomes? AA-tRNA no NNN UUU AAA CCCPhe-tRNA 0.34 1.56 0.20 0.30Lys-tRNA 0.80 0.56 6.13 0.60Pro-tRNA 0.24 0.20 0.18 0.73pmoles AA-tRNA bound with:Features of the Genetic CodeTable 3.4.4 The Genetic CodePosition in Codon1st 2nd . 3rdU C A G U UUU Phe UCU Ser UAU Tyr UGU Cys UUUC Phe UCC Ser UAC Tyr UGC Cys CUUA Leu UCA Ser UAA Term UGA Term AUUG Leu UCG Ser UAG Term UGG Trp GC CUU Leu CCU Pro CAU His CGU Arg UCUC Leu CCC Pro CAC His CGC Arg CCUA Leu CCA Pro CAA Gln CGA Arg ACUG Leu CCG Pro CAG Gln CGG Arg GA AUU Ile ACU Thr AAU Asn AGU Ser UAUC Ile ACC Thr AAC Asn AGC Ser CAUA Ile ACA Thr AAA Lys AGA Arg AAUG* Met ACG Thr AAG Lys AGG Arg GG GUU Val GCU Ala GAU Asp GGU Gly UGUC Val GCC Ala GAC Asp GGC Gly CGUA Val GCA Ala GAA Glu GGA Gly AGUG* Val GCG Ala GAG Glu GGG Gly G* Sometimes used as initiator codons.Degeneracy of the code•Degeneracy refers to the fact that almost all amino acids are encoded by multiple codons.•Degeneracy is found primarily in the 3rd position of the codon, i.e. the nucleotide in the 3rd position can change without changing the amino acid specified.•In some cases (Leu and Arg), the 1st position can also be degenerate.Groupings of codons•Of the 64 codons, 61 specify amino acids and the other 3 are signals to terminate translation• 9 codon families. –E.g. encode Thr with•ACU•ACC•ACA•ACG•13 codon pairs –E.g. encode Asp with: Glu with •GAUGAA•GACGAGCodons for initiation of translation•Major codon for initiation is AUG•Regardless of codon used, the first amino acid incorporated in E. coli is formyl-Met.•For the 4288 genes identified in E. coli:AUG is used for 3542 genes.GUG is used for 612 genes.UUG is used for 130 genes.AUU is used for 1 gene.CUG may be used for 1 gene.Codons for termination of translation•UAA, UAG, UGA•For the genes identified in E. coli:UAA is used for 2705 genes.UGA is used for 1257 genes.UAG is used for 326 genes.Genetic code is univeral (almost)•All organisms so far examined use the code as originally deduced (or something very close to it).•The rare exceptions involve limited differences.–e.g. In RNA derived from mitochondrial DNA, UGA encodes Trp instead of serving as a stop codon.–Thus UGA and UGG form a codon pair in this case.Differential codon usage•Some codons are used much more frequently than others to encode a particular amino acid.•The pattern of codon usage varies between species and even among tissues within a species.•Correlates with tRNA abundance.•Pattern of codon usage can be a predictor of level of expression of a gene.•Preferred codon usage is a help in reverse genetics.•Some nucleotides in the 1st position of the anticodon (in tRNA) can