Slow peptide bond formation by proline and other N-alkylamino acids in translation Michael Y. Pavlova, Richard E. Wattsb, Zhongping Tanc,1, Virginia W. Cornishc, Måns Ehrenberga,2, and Anthony C. Forsterb aDepartment of Cell and Molecular Biology, Uppsala University, BMC, Box 596, 751 24 Uppsala, Sweden; bDepartment of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, 2222 Pierce Avenue, Nashville, TN 37232; and cDepartment of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027 Edited by Dieter So¨ll, Yale University, New Haven, CT, and approved November 17, 2008 (received for review September 15, 2008) Proteins are made from 19 aa and, curiously, one N-alkylamino acid synthesized N-NitroVeratrylOxyCarbonyl (NVOC)-amino- (‘‘imino acid’’), proline (Pro). Pro is thought to be incorporated by acyl-pdCpA; the amino protecting group (NVOC) is then the translation apparatus at the same rate as the 19 aa, even removed by photolysis (15). We first need to ascertain the though the alkyl group in Pro resides directly on the nitrogen effect of lack of nucleoside modifications and of a penultimate nucleophile involved in peptide bond formation. Here, by combin- dC, in those aminoacyl-tRNA constructs (Fig. 1 Upper)on ing quench-flow kinetics and charging of tRNAs with cognate and translation kinetics. Posttranscriptional modifications have noncognate amino acids, we find that Pro incorporates in transla- been shown to affect marginally translation kinetics in the case tion significantly more slowly than Phe or Ala and that other of one tRNA, E. coli tRNAPheC3G-G70C, at least for N-acetyl- N-alkylamino acids incorporate much more slowly. Our results Phe-Phe dipeptide synthesis from poly(U) template at 5 °C, show that the slowest step in incorporation of N-alkylamino acids which proceeded with a codon translation time of Ϸ1 s (16). is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. To assess the effects of lack of modifications also at near in The relative incorporation rates correlate with expectations from vivo conditions (37 °C and codon translation times faster than organic chemistry, suggesting that amino acid sterics and basicities 45 ms) we employ a version of our purified translation system affect translation rates at the peptidyl transfer step. Cognate optimized for speed and accuracy at 37 °C (17, 18). The kinetics of isoacceptor tRNAs speed Pro incorporation to rates compatible ribosomal synthesis of [3H]fMet-Phe-tRNAPheC3G-G70C from with in vivo, although still 3–6 times slower than Phe incorporation [3H]fMet-tRNAfMet and Phe-tRNAPheC3G-G70C, prepared by li- from Phe-tRNAPhe depending on the Pro codon. Results suggest gation and photodeprotection (termed Phe-tRNAPheB; Fig. 1) that Pro is the only N-alkylamino acid in the genetic code because are first compared with the corresponding kinetics for the native it has a privileged cyclic structure that is more reactive than other tRNAPhe charged with Phe by Phe-tRNA synthetase. We mea- N-alkylamino acids. Our data on the variation of the rate of sure the average times, ␶, for both chemical reactions of codon Pro incorporation of Pro from native Pro-tRNA isoacceptors at 4 translation, GTP hydrolysis on EF-Tu (␶GTP; discussed below) different Pro codons help explain codon bias not accounted for by and overall dipeptide synthesis (␶dip), in the same reaction the ‘‘tRNA abundance’’ hypothesis. mixtures. The similar and short translation times for Phe- tRNAPheB and native Phe-tRNAPhe [Fig. 2 A and B, Table 1, and non-natural amino acids ͉ ribosomes ͉ tRNA ͉ EF-Tu ͉ GTPase supporting information (SI) Table S1] show that the penultimate dC and the absence of all 10 posttranscriptional modifications in Phe -alkylation confers special properties on amino acids and tRNA (Fig. 1) have only a minor effect on dipeptide synthesis polypeptides. The N-alkyl group in Pro-tRNAPro resides kinetics, which also validates our combination of methods. N Next, we measured the effect on translation of changing the directly on the nitrogen nucleophile that forms the peptide bond PheB in translation, yet this is somehow tolerated by the translation cognate amino acid on tRNA , Phe, to a much smaller, apparatus. N-alkyl groups in polypeptides and proteins kink noncognate, natural amino acid, Ala. Again, the effect is minor 3-dimensional structures, result in slow isomerization between (Fig. 2 B and C and Table 1). cis and trans conformations in the case of Pro, and can improve In marked contrast, changing the cognate Phe to its simplest N-alkyl analog, N-methyl-Phe (Fig. 1), increases the average the pharmacological properties of peptides by decreasing their ␶ degradation by proteases and increasing their membrane per- dipeptide synthesis time ( dip) by a factor of 8,000 to 3.5 min (Fig. 2D and Table 1). Nevertheless, virtually all N-methyl-Phe- meability. To investigate these special properties or endow them PheB PheB on translation products, there have been many studies incorpo- tRNA was converted to fMet-N-methyl-Phe-tRNA rating N-alkylamino acids (‘‘imino acids’’) in translation (1–13). within 30 min (Fig. 2D). Such a huge inhibitory effect of a small Small linear N-alkylamino acids (Fig. 1 Upper Right), because of methyl group on the kinetics of dipeptide formation is surprising, their similarity to Pro, were expected to incorporate well in albeit consistent with our previous inability to saturate incor- translation, but the efficiencies were variable (1–13). Incomplete poration at the third position of a tetrapeptide with this substrate incorporations in purified translation systems (8–13) are partic- (11). Mechanistic insight into the remarkably slow incorporation ularly puzzling because of the lack of competing reactions, incubation times of tens of minutes, and an average codon Author contributions: M.Y.P., M.E., and A.C.F. designed research; M.Y.P. performed re- translation time comparable with that in Escherichia coli (45 ms) search; M.Y.P., R.E.W., Z.T., V.W.C., and A.C.F. contributed new reagents/analytic tools; (14). Here, we investigate the kinetics of incorporation of Pro M.Y.P., M.E., and A.C.F. analyzed data; and A.C.F. wrote the paper. and other N-alkylamino acids by combining quench-flow trans- The authors declare no conflict of interest. lation kinetics and charging of tRNAs with noncognate/ This article is a PNAS Direct Submission. unnatural amino acids (Fig. 1). 1Present address: Laboratory for Bioorganic Chemistry, Sloan–Kettering Institute, 1275 York Avenue, New York, NY 10065. Results 2To whom correspondence should be addressed. E-mail: [email protected]. A highly flexible method for charging tRNAs with noncognate This article contains supporting information online at www.pnas.org/cgi/content/full/ amino acids and N-alkylamino acids is to use T4 RNA ligase 0809211106/DCSupplemental. Ϫ to join an unmodified tRNA CA transcript to a chemically © 2008 by The National Academy of Sciences of the USA 50–54 ͉ PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809211106 Downloaded by guest on September 29, 2021 BIOCHEMISTRY 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. of N-methyl-Phe comes from measuring ␶GTP, the average time the ribosomal A site. ␶GTP is only 4 times longer for N-methyl- for GTP hydrolysis on elongation factor Tu (EF-Tu). GTP Phe-tRNAPheB (Fig. 2D and Table 1) than for Phe-tRNAPheB. hydrolysis is greatly stimulated when the ternary complex of Further, N-methyl-Phe incorporation is strictly dependent on the aminoacyl-tRNA–EF-Tu–GTP is bound to a cognate codon at aminoacyl-tRNA carrier, EF-Tu (Fig. S1), excluding the possi- Table 1. Average times for dipeptide synthesis from fMet-tRNAfMet and different aminoacyl-tRNAs Aminoacyl-tRNA Codon ␶dip,ms ␶GTP,ms ␶acc,pep,ms Phe-tRNAPhe UUU 19.6 (Ϯ2.1) 7.8 (Ϯ0.6) 11.9 (Ϯ2.1) Phe-tRNAPhe UUC 18.5 (Ϯ1.7) 7.7 (Ϯ0.6) 10.8 (Ϯ1.8) Phe-tRNABulk UUC 20.4 (Ϯ1.7) 8.9 (Ϯ0.8) 11.5 (Ϯ1.9) Phe-tRNAPheB UUC 24.4 (Ϯ2.3) 13.0 (Ϯ0.7) 11.4 (Ϯ2.4) N-methyl-Phe-tRNAPheB UUC 200,000 (Ϯ12,000) 52.6 (Ϯ7.3) 199,900 (Ϯ12,000) N-butyl-Phe-tRNAPheB UUC Undetectable 67.7 (Ϯ27) Undetectable Ala-tRNAPheB UUC 37.0 (Ϯ5.1) 13.2 (Ϯ1.4) 23.9 (Ϯ7.1) Pro-tRNAPheB UUC 571.3 (Ϯ65) 16.1 (Ϯ3.2) 555 (Ϯ65) Pro-tRNABulk CCG 62.5 (Ϯ7.6) 12.5 (Ϯ1.4) 50.0 (Ϯ7.7) Pro-tRNABulk CCA 66.7 (Ϯ8.9) 18.9 (Ϯ2.8) 47.8 (Ϯ9.3) Pro-tRNABulk CCU 71.4 (Ϯ9.7) 23.8 (Ϯ3.5) 47.6 (Ϯ10.3) Pro-tRNABulk CCC 116.3 (Ϯ16.1) 50.0 (Ϯ5.1) 66.3 (Ϯ16.9) Data are expressed as average times for GTP hydrolysis (␶GTP) and dipeptide synthesis (␶dip) measured in the same reaction mixture. The average time for accommodation/peptidyl transfer is calculated as ␶acc,pep ϭ ␶dip Ϫ ␶GTP. All experiments were conducted at 37 °C in high-accuracy polyamine containing LS3 buffer (see Materials and Methods); each experiment was performed at least twice.