Orientation of the tRNA anticodon in the ribosomal P-site: Quantitative footprinting with U₃₃-modified, anticodon stem and loop domains

Abstract

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA–ribosomal RNA (rRNA) interactions. To better understand these interactions, U₃₃-substituted yeast tRNA^Phe anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (K_p) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA^Phe, ASL-U₃₃, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m³U₃₃, ASL-C₃₃, and ASL-dU₃₃, respectively) were compared. Yeast tRNA^Phe and the ASL-U₃₃ protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA^Phe enhanced protection of C1400, but the ASL-U₃₃ and U₃₃-substituted ASLs did not. Two residues, G926 and G1338 with K_ps ≈ 50–60 nM, were afforded significantly greater protection by both yeast tRNA^Phe and the ASL-U₃₃ than other residues, such as A532, A794, C795, and A1339 (K_ps ≈ 100–200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U₃₃-substituted ASLs as compared with that of the ASL-U₃₃. ASL-m³U₃₃ and ASL-C₃₃ protected G530, A532, A794, C795, and A1339 as well as the ASL-U₃₃. However, protection of G926 and G1338 (K_ps between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U₃₃ (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU₃₃ were reduced as compared to that of the ASL-U₃₃, a proportionally greater reduction of G926 and G1338 protections was observed (K_ps = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U₃₃ of tRNA are positioned close to each other.