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

Abstract

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been 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 or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th 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 site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(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 tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two 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, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A 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.

tRNA Thiolated Pyrimidines as Targets for Near‐Ultraviolet‐Induced Synthesis of Guanosine Tetraphosphate in Escherichia coli

“…It is not yet known whether it is unique or whether other near-ultraviolet-absorbing residues are involved. A likely candidate could be the 5-carbomethoxy-uracil residue present in the anticodon loop of tRNAva' and tRNAser and which is known to be capable of photoactivation by near-ultraviolet light [31].…”

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

“…Reconstituted 30S subunits were purified on sucrose gradients, programmed with the T4 gene32 mRNA fragment, and allowed to bind 3Ј-biotinylated tRNA Val1 in the P-site. The complex was irradiated with UV light to form a site-specific cross-link between the tRNA Val1 and 16S rRNA in the 30S subunit (Ofengand et al 1979;Prince et al 1982). The 16S rRNA cross-linked to 3Ј-biotin-tRNA Val1 was extracted and purified by binding to magnetic streptavidin beads (von Ahsen and Noller 1995).…”

A General Secondary‐Structure Model for Procaryotic and Eucaryotic RNAs of the Small Ribosomal Subunits

“…Indeed, there is strong evidence that both the central and 3'-terminal domains contain sequences that are exposed on the surface of the subunit and located at the interface of the two subunits in the E. coli ribosome [18,27,281, some of which make contact with the large subunit [29]. Furthermore, one of these highly conserved regions in the 3'-domain of the E. coli RNA appears to be in contact with the anticodon of the tRNA (C. Ehresmann, R. Millon, J. Ofengand and B. Ehresmann, unpublished results) when it is bound at the ribosomal P site [31].…”