translation zamecnik 1957 first in vitro protein synthesis requires gtp, soluble proteins,...
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TranslationZamecnik 1957First in vitro protein synthesisRequires GTP, soluble proteins, ribosomes, charged aminoacyl tRNAs,
mRNA
Protein synthesis catalyzed on ribosome (proteins + RNAs)20,000 ribosomes in bacterial cellOnly 1 polypeptide synthesized on ribosome at one timeRibosome catalyzes peptide bond formation between an aminoacyl
tRNA and a peptidyl tRNA~20 amino acids incorporated per secondSequence 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
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RNA metabolismTranslationCracking the genetic code1. Experiments showed that each amino acid is coded by 3 nucleotides termed a codon42 = 16 combinations, 43 = 64 combinationsNirenberg and Matthaei 1961Synthetic poly(U) + E.Coli extract, GTP, ATP, 20 radioactive AAs in separate tubes radioactive polypeptide with only PheSame exp. with poly(C) radioactive polypeptide with only ProSame exp. with poly(A) radioactive polypeptide with only LysSame exp. with poly(G) aggregates
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RNA metabolismTranslationCracking the genetic codeKhorana 1960sTermination (stop) codons: UAG amber
UAA ochreUGA opal
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RNA metabolismTranslationCracking the genetic code2. Genetic code is nonoverlapping
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RNA metabolismTranslationCracking the genetic code
C
G
A U
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RNA metabolismTranslationCracking the genetic code3. The code is read in a sequential manner starting from a fixed point
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RNA metabolismTranslationCracking the genetic code4. The code is degenerate
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TranslationCracking the genetic code4. The code is degenerate
RNA metabolism
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TranslationWobble hypothesis1. First two bases of mRNA form strong Watson-Crick bp with tRNA2. First base of anticodon determines # codons recognized by tRNA
RNA metabolism
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Translation
RNA metabolism
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RNA metabolismTranslationRibosomes
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RNA metabolismTranslationRibosomes
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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 SynthesisPoul Nissen, Jeffrey Hansen, Nenad Ban, Peter Moore, Thomas SteitzScience, 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)
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TranslationrRNA
RNA metabolism
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RNA metabolismTranslationtRNA
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RNA metabolism TranslationActivation of amino acids
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RNA metabolism
TranslationInitiation by a specific amino acid
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RNA metabolismTranslationInitiation 5’-AUG position relative to Shine-Dalgarno sequence
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RNA metabolism
TranslationInitiation
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RNA metabolism
TranslationElongation - step 1Bind 2nd aminoacyl-tRNA
AA2
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RNA metabolism
TranslationElongation - step 2Form 1st peptide bond
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RNA metabolism
TranslationElongation - step 3Translocation
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TranslationTermination
RNA metabolism
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TranslationPolysome
RNA metabolism
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TranslationCoupled transcription-translation in proks
RNA metabolism
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TranslationInhibitors of bacterial protein synthesis = antibiotics
RNA metabolism
This end cannot be used to add another AA onto the peptide chain
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TranslationInhibitors of protein synthesis = antibiotics
RNA metabolism
Inhibits peptidyl transfer
Inhibits binding of aminoacyl-tRNA
Misreads genetic code & inhibits initiation