Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates.
The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp >> Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.
Wohlgemuth
Ingo
I
Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Strasse 10, D-58448 Witten, Germany.
Brenner
Sibylle
S
Beringer
Malte
M
Rodnina
Marina V
MV
D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics
D000596:Amino Acids; D003062:Codon; D010455:Peptides; D016337:RNA, Catalytic; D012343:RNA, Transfer; D011392:Proline