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

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

RNA metabolismTranslationCracking the genetic codeKhorana 1960sTermination (stop) codons: UAG amber

UAA ochreUGA opal

RNA metabolismTranslationCracking the genetic code2. Genetic code is nonoverlapping

RNA metabolismTranslationCracking the genetic code

C

G

A U

RNA metabolismTranslationCracking the genetic code3. The code is read in a sequential manner starting from a fixed point

RNA metabolismTranslationCracking the genetic code4. The code is degenerate

TranslationCracking the genetic code4. The code is degenerate

RNA metabolism

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

Translation

RNA metabolism

RNA metabolismTranslationRibosomes

RNA metabolismTranslationRibosomes

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)

TranslationrRNA

RNA metabolism

RNA metabolismTranslationtRNA

RNA metabolism TranslationActivation of amino acids

RNA metabolism

TranslationInitiation by a specific amino acid

RNA metabolismTranslationInitiation 5’-AUG position relative to Shine-Dalgarno sequence

RNA metabolism

TranslationInitiation

RNA metabolism

TranslationElongation - step 1Bind 2nd aminoacyl-tRNA

AA2

RNA metabolism

TranslationElongation - step 2Form 1st peptide bond

RNA metabolism

TranslationElongation - step 3Translocation

TranslationTermination

RNA metabolism

TranslationPolysome

RNA metabolism

TranslationCoupled transcription-translation in proks

RNA metabolism

TranslationInhibitors of bacterial protein synthesis = antibiotics

RNA metabolism

This end cannot be used to add another AA onto the peptide chain

TranslationInhibitors of protein synthesis = antibiotics

RNA metabolism

Inhibits peptidyl transfer

Inhibits binding of aminoacyl-tRNA

Misreads genetic code & inhibits initiation