Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

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Abstract
The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of E. coli ribosomes upon near-UV irradiation previously reported was studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310-335 nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence of absence of 4-thiouridine in the tRNA. The kinetics of cross-liking showed single-hit behavior. Twofold more cross-linking was obtained with tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P [peptidyl] site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, Ar, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of poly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe or tRNAfMet could not. By sequence comparison of the reactive and nonreactive tRNA, the site of attachment in the tRNA was deduced to be the 5''-anticodon base, cmo5U, or mo5U in all of the reactive tRNA. The attachment site in 16S RNA is described elsewhere. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most 2 nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, < 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. When the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A [aminoacyl] site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.