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Gene expression DNA ------------> RNA ------------> Transcription RNA processing ----------->Protein Translation 1) 5’ capping 2) 3’ Poly A tails splicing 29 Gene expression DNA ------------> RNA ------------> Transcription RNA processing ----------->Protein Translation 1) 5’ capping 2) 3’ Poly A tails splicing 29 Translation Bacterial mRNA Ribosome Binding site Coding sequence Determines seq of amino acid Start codon Stop codon Fig 12.11 30 Translation Codon: A sequence of 3 nucleotides that specifies a particular amino acid Genetic code is Degenerate (=more than one codon specifies a particular amino acid 31 Discovery of The Genetic Code Use of synthetic RNA AAAAAAAAAAAAAAA => Lys-Lys-Lys-Lys-Lys UUUUUUUUUUUUUUU => Phe-Phe-Phe-Phe-Phe 15 NT => 5 a.a 3 NT / 1 a.a 32 Discovery of The Genetic Code 1) Add RNA triplet : e.g., CCA Ala*+ 19 other aa Leu* 19 other aa pro *+ 19 other aa 2) Add mixture of 20 tRNA(next page ) attached to each a.a with only one a.a being radioactively labeled +ribosome tRNA Specialized RNA that functions in transferring an amino acid to growing peptide sequence Structure of tRNA 3’ single stranded region Amino acid attach site Fig 12.14 Anti-codon Complementary to codon sequence CCA G G U 34 Translation 3) Isolate tRNA bound ribosome by filtering 4) Determine which tube has radioactivity CCA codes for proline Ala*+ 19 other aa Leu* 19 other aa pro *+ 19 other aa Translation 37 Translation mRNA, tRNA, ribosomes, translation factors 38 tRNA Fig 12.14 Structure of tRNA 3’ single stranded region Amino acid attach site Anti-codon Complementary to codon sequence 39 Aminoacyl-tRNA -synthetase Fig 12.15 - Catalyzes the attachment of amino acids to tRNA by using ATP - 20 types of amino acyl-tRNA synthetase exits (one for each a.a) 1) Binding of Amino acid & & ATP a.a is tansfered to tRNA Charged tRNA Aminoacyl-tRNA -synthetase 40 Ribosome Fig 12.16 Large subunit Exit site for tRNA E site P site Actually synthesis Of a.a chain A site Charged tRNA come in small subunit 41 3 stages of translation Initiation Elongation Termination 42 3 stages of translation Initiation 1) Small subunit binds to mRNA 2) Initiator tRNA comes in 3) Large subunit comes in Initiator tRNA mRNA Use GTP as energy source Fig 12.18 43 3 stages of translation Initiator tRNA mRNA Initiation Elongation Synthesis from start codon to stop codon Termination Disassembly of the complex at stop codon releasing Polypeptide Fig 12.18 44 Translation 45 Gene regulation DNA ------------> RNA ------------> Transcription RNA processing ----------->Protein Translation 1) 5’ capping 2) 3’ Poly A tails splicing 46 Gene regulation at the level of translation Example Iron toxicity in mammals Iron is a vital cofactor for many cellular enzymes However, it is toxic at high levels Ferritin : protein that can bind to excess iron When there is high iron => Hight ferritin 47 Gene regulation at the level of translation Iron toxicity in mammals Low iron in cytosol IRP (iron regulatory protein) binds to IRE ferritin mRNA Inhibition of ferritin translation high iron in cytosol Iron binds to IRP=>inhibit binding of IRP to IRE ferritin translation Fig 13.22 48 Gene regulation DNA ------------> RNA ------------> Transcription RNA processing ----------->Protein Translation Point of regulation 49