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. 2009 Jan 6;106(1):50-4.
doi: 10.1073/pnas.0809211106. Epub 2008 Dec 22.

Slow peptide bond formation by proline and other N-alkylamino acids in translation

Michael Y Pavlov  1 , Richard E Watts, Zhongping Tan, Virginia W Cornish, Måns Ehrenberg, Anthony C Forster
Affiliations

Slow peptide bond formation by proline and other N-alkylamino acids in translation

Michael Y Pavlov et al. Proc Natl Acad Sci U S A. .

Abstract

Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Combination of tRNAs charged with noncognate/unnatural amino acids and quench-flow translation kinetics. (Upper) The 5 amino- and N-alkylamino acids used in this study, one of which is shown charged on tRNAPheB. This synthetic, unmodified tRNA is based on natural E. coli tRNAPhe (black with purple anticodon; posttranscriptional modifications are in green), with changes in blue (11). (Lower) Aminoacyl-tRNA prebound to elongation factor Tu–GTP is mixed with ribosomes preinitiated with fMet-tRNAinitiator and then quenched rapidly with formic acid. Reactants are in green; products measured are in red.
Fig. 2.
Fig. 2.
Kinetics of dipeptide synthesis with aminoacyl- and N-alkylaminoacyl-tRNASPhe. Kinetics of dipeptide synthesis (▴) from fMet-tRNAifMet and from Phe-tRNAPhe (A), Phe-tRNAPheB (B), Ala-tRNAPheB (C), N-methyl-Phe-tRNAPheB (D), N-butyl-Phe-tRNAPheB (E), and Pro-tRNAPheB (F) are shown. Kinetics of the prerequisite, faster GTP hydrolysis (▵) by EF-Tu in the same reactions are shown. The ribosome had the Phe codon UUC at the A site. All kinetics were measured at 37 °C in high-accuracy polyamine containing LS3 buffer (see Materials and Methods). Representative plots are shown for each assay.
Fig. 3.
Fig. 3.
Kinetics of dipeptide synthesis with the native Pro-tRNAPro isoacceptors. (A) Sequences of the 3 E. coli tRNAPro isoacceptors. Nonconserved sequences are in red, and anticodons are underlined. The measured number of molecules per E. coli cell (28) and accepted codon specificities are shown (the posttranscriptional modifications have not been reported, but the Pro-1 anticodon is accepted to be modified at U34 to cmo5U) (31). (B) Lack of correlation between abundance of Pro codons in E. coli genes and measured number of cognate isoacceptor tRNAs. (C–H) Kinetics of dipeptide synthesis (▴) and accompanying GTP hydrolysis (▵) from native isoacceptors at all 6 codons for Phe and Pro. All kinetics were measured at 37 °C in high-accuracy polyamine containing LS3 buffer (see Materials and Methods). Representative plots are shown for each assay.
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