Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Kinetics of the rRNA-Catalyzed Peptidyl-Transfer to Native aa-tRNAs

High levels of accuracy in transcription, aminoacylation, and mRNA translation are essential for all life forms. However, a very high accuracy level may be incompatible with a high rate of protein synthesis. For maximal rate of cell growth there is therefore an optimal balance between accuracy and rate of protein elongation. We have used an in vitro system, optimized for high rate and accuracy in protein elongation, to characterize the reaction between wild-type E. coli ribosomes in post-translocation state and cognate as well as near-cognate ternary complexes (1). At 37 °C, we estimate the maximal rate (kcat) of fMet-Phe dipeptide formation as 130 s-1. Under the same condition, we estimate the near-cognate missense error as 3·10-7. The kcat-value is compatible with the average in vivo protein elongation rate, including the translocation step, estimated as 22 amino acids per second per ribosome for E. coli bacteria growing in rich medium at 37 °C. Our in vitro estimate for the missense error level is, at the same time, on the lower side of the ?consensus? estimate of about one missense substitution per three thousand incorporated amino acids. By determining the temperature dependence of the rate limiting step subsequent to GTP hydrolysis we estimated the activation enthalpy (ΔH‡) and entropy (TΔS‡) of this step as 17 kcal·mol-1 and 2 kcal·mol-1, respectively. These values are in good agreement with previous theoretical as well as experimental estimates of the activation free energy of the ribosome-catalyzed peptidyl-transfer reaction. Therefore our data suggest, but do not prove, that under optimal experimental conditions peptidyl-transfer itself, rather than tRNA accommodation, is rate limiting in the sequence of chemical events that leads from ternary complex association with the A site to peptide bond formation. Our more recent data show a clear pH dependence of the overall rate of peptidyl transfer for several different aa-tRNAs, in line with the prevailing model for RNA catalyzed peptidyl-transfer.

References and Footnotes
  1. Johansson et al. Mol Cell 30, 589-598 (2008).

Magnus Johansson1
Kaweng Ieong1
Elli Bouakaz1
Martin Lovmar1
Peter Strazewski2
Michael Pavlov1
Måns Ehrenberg1*

1Dept of Cell and Molecular Biology
BMC, Uppsala University
Box 596
S-751 24 Uppsala
2Lab de Synthèse de Biomolécules
Institut de Chimie et Biochimie Moléculaires et
Supramoléculaires (UMR 5246)
Université Claude Bernard
Lyon 1, France

*Phone: +46 18 471 4213
Fax: +46 18 471 4262