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

  • S. SALMAN ASHRAF (a1) (a2), RICHARD GUENTHER (a1) and PAUL F. AGRIS (a1)

Abstract

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA–ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNAPhe 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 (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNAPhe, 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 tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNAPhe enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with Kps ≈ 50–60 nM, were afforded significantly greater protection by both yeast tRNAPhe and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (Kps ≈ 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 (Kps 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 (Kps = 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.

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Reprint requests to: Paul F. Agris, 128 Polk Hall, Department of Biochemistry, NCSU Box 7622, North Carolina State University, Raleigh, North Carolina 27695-7622, USA; e-mail: agris@bchserver.bch.ncsu.edu.

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

  • S. SALMAN ASHRAF (a1) (a2), RICHARD GUENTHER (a1) and PAUL F. AGRIS (a1)

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