1570 Nucleic Acids Research, 2006, Vol. 34, No. 5 tRNAArg4, tRNAAsn, tRNAAsp1, tRNAHis, tRNALeu3, tRNAPhe, tRNAThr4 and tRNAVal1 (data not shown). Similar smears located above tRNA bands have been taken as evidence of p-tRNA heterogeneity in previous studies (16). However, since the p-tRNALys species considered in the present study were only those recovered in the aqueous phase of the phenol extract and those that migrated close to the free and aminoa- cylated tRNA forms in acid gel electrophoresis, it may be assumed that they carried peptide chains containing only a few amino acid residues (17). Presumably, those p-tRNAs in which the protein nature of the peptide predominates over the RNA character of the molecule are not present in the aqueous phase but precipitate with protein during phenol extraction (17). Why is p-tRNALys most rapidly accumulated upon transfer of the pth(Ts) mutant to 40 C (4)? The propensity of p-tRNALys to drop-off readily either upon expression of a minigene (12) or of a gene in which a lysine codon precedes the termination triplet (18), may be ascribed mainly to the long pause at the termination codon before the ribosome terminates (18). However, the soluble heterogeneous p-tRNALys that accumulated in the presence of limited levels of Pth(Ts) protein could result from drop-off at the lysine codons frequently located in the initial open reading frame positions of E.coli genes (19). Based on the relative high frequency of the AAA codon at positions 2, 3, 4 and 5 in the E.coli reading frames (19), we favour that the heterogeneous and short p-tRNALys species originate by drop-off early after translation initiation of bacterial mRNAs. In contrast, the infrequent loca- tion of glycine and cysteine codons at early positions in the reading frames, correlates with the slow accumulation of p-tRNAGly and p-tRNACys (19,4). This proposal is consistent with observations, namely, that the propensity of the p-tRNAs to drop-off correlates inversely with the length of the coding sequence as the oligopeptidyl moiety increases in size from 2 to 6 amino acid residues (20), that drop-off occurs irrespective of whether the codon in the ribosomal A-site is a sense or stop codon (21). It is also possible that a variety of minigenes containing lysine codons exist in the bacterial chromosome the expression of which would result in the accu- mulation of heterogeneous p-tRNALys. Further experiments designed to decide which, if either, of these hypotheses is correct are required in order to establish the origin of the accumulated p-tRNALys in the pth(Ts) cells incubated at non-permissive temperatures. ACKNOWLEDGEMENTS The authors wish to thank Richard Buckingham for providing plasmid constructs, Marco A. Magos-Castro, Jose´ G. Bueno-Mart´ınez and Gloria Leo´ n Avila for technical support and an anonymous referee for critical review and comments on the manuscript. This work was supported by grants from the Consejo Nacional de Ciencia y Tecnolog´ıa (CONACyT, Mexico) to G.G. One of us (S.V.D) was supported by grants from CONACyT and Consejo del Sistema Nacional de Educacio´ n Technolo´ gica (COSNET). Funding to pay the Open Access publication charges for this article was provided by Centro de Investigacio´ n y de Estudios Avanzados. Conflict of interest statement. None declared. REFERENCES 1. Atherly,A.G. and Menninger,J.R. (1972) Mutant E.coli strain with temperature sensitive peptidyl-transfer RNA hydrolase. Nature New Biol., 240, 245–246. 2. Garc´ıa-Villegas,M.R., De la Vega,F.M., Galindo,J.M., Segura,M., Buckingham,R.H. and Guarneros,G. (1991) Peptidyl-tRNA hydrolase is involved in inhibition of host protein l synthesis. EMBO J., 10, 3549–3555. 3. 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