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

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.non-natural amino acids ͉ ribosomes ͉ tRNA ͉ EF-Tu ͉ GTPase

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

“…This final step in decoding encompasses the release of both inorganic phosphate and Val-tRNA Val from EF-Tu•GDP, the accommodation of the Val-tRNA Val into the A site and the formation of the peptide bond. It remains uncertain which one of these substeps limits the value of k pep for wild-type aa-tRNAs, and it may depend upon the assay conditions and the aa-tRNA that are used (28,31,32). Here k pep is measured with ternary complexes containing 3′-[ 32 P]-labeled Val-tRNA Val using a P1 nuclease assay (33).…”

Mg 2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide

“…1). A stretch of three Pro residues could constitute a ribosome-stalling motif sequence (16)(17)(18)(19). Although mgtA-lacZ expression in the wildtype strain was up-regulated sevenfold upon decreasing [Mg 2+ ] from 1.6 mM (high) to 0.016 mM (low), the A86C mutant exhibited a 13-fold increased expression of the mgtA-lacZ fusion at high Mg 2+ , compared with the wild type (Fig.…”

“…These findings suggest that the codon composition of the mgtL + sequence slows down translation just enough to make stalling exquisitely dependent on the Mg 2+ concentration. Incorporation of Pro or other N-alkyl amino acids is an impediment to rapid translation (17). Nascent peptides containing stretches of Pro codons can induce ribosome stalling (18), and translation of the CCC and CCU Pro codons is particularly prone to +1-frameshifting (25).…”

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

“…25 %), the yield of H16Q48 was approximately half (Figure 1 f). We attribute this lower efficiency to the intercalated position of the glutamine between two prolines (Figure 1 a), which present the slowest translational velocity among the 20 natural amino acids 21…”