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RNA metabolism Translation Zamecnik 1957 First in vitro protein synthesis Requires GTP, soluble proteins, ribosomes, charged aminoacyl tRNAs, mRNA Protein synthesis catalyzed on ribosome (proteins + RNAs) 20,000 ribosomes in bacterial cell Only 1 polypeptide synthesized on ribosome at one time Ribosome catalyzes peptide bond formation between an aminoacyl tRNA and a peptidyl tRNA ~20 amino acids incorporated per second Sequence of peptide determined by order of codons within mRNA (protein made from N- to C-terminus) Multiple ribosomes can be simultaneously translating off same mRNA (polyribosome) Transcription and translation are coupled in prokaryotes RNA metabolism Translation Cracking the genetic code 1. Experiments showed that each amino acid is coded by 3 nucleotides termed a codon 42 = 16 combinations, 43 = 64 combinations Nirenberg and Matthaei 1961 Synthetic poly(U) + E.Coli extract, GTP, ATP, 20 radioactive AAs in separate tubes radioactive polypeptide with only Phe Same exp. with poly(C) radioactive polypeptide with only Pro Same exp. with poly(A) radioactive polypeptide with only Lys Same exp. with poly(G) aggregates RNA metabolism Translation Cracking the genetic code Khorana 1960s Termination (stop) codons: UAG amber UAA ochre UGA opal RNA metabolism Translation Cracking the genetic code 2. Genetic code is nonoverlapping RNA metabolism Translation Cracking the genetic code C G A U RNA metabolism Translation Cracking the genetic code 3. The code is read in a sequential manner starting from a fixed point RNA metabolism Translation Cracking the genetic code 4. The code is degenerate RNA metabolism Translation Cracking the genetic code 4. The code is degenerate RNA metabolism Translation Wobble hypothesis 1. First two bases of mRNA form strong Watson-Crick bp with tRNA 2. First base of anticodon determines # codons recognized by tRNA RNA metabolism Translation Translation Ribosomes RNA metabolism Translation Ribosomes RNA metabolism The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution. Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter Moore, Thomas Steitz. Science, Vol. 289 (2000) The Structural Basis of Ribosome Activity in Peptide Bond Synthesis Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter Moore, Thomas Steitz Science, Vol. 289 (2000) A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits Schmeing TM, Seila AC, Hansen JL, Freeborn B, Soukup JK, Scaringe SA, Strobel SA, Moore PB, Steitz TA. Nature Structural Biology, Vol. 9 (2002) Translation rRNA RNA metabolism Translation tRNA RNA metabolism RNA metabolism Translation Activation of amino acids RNA metabolism Translation Initiation by a specific amino acid RNA metabolism Translation Initiation 5’-AUG position relative to Shine-Dalgarno sequence RNA metabolism Translation Initiation RNA metabolism Translation Elongation - step 1 Bind 2nd aminoacyl-tRNA AA2 RNA metabolism Translation Elongation - step 2 Form 1st peptide bond RNA metabolism Translation Elongation - step 3 Translocation RNA metabolism Translation Termination Translation Polysome RNA metabolism RNA metabolism Translation Coupled transcription-translation in proks RNA metabolism Translation Inhibitors of bacterial protein synthesis = antibiotics This end cannot be used to add another AA onto the peptide chain RNA metabolism Translation Inhibitors of protein synthesis = antibiotics Inhibits peptidyl transfer Inhibits binding of aminoacyl-tRNA Misreads genetic code & inhibits initiation
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