Ribosome-Catalyzed Peptide-Bond Formation with an A-Site Substrate Covalently Linked to 23 S Ribosomal RNA-Reference-Cited by-同舟云学术

Ribosome-Catalyzed Peptide-Bond Formation with an A-Site Substrate Covalently Linked to 23 S Ribosomal RNA

Published:1998-04-10 Issue:5361 Volume:280 Page:286-289
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Green Rachel¹²,Switzer Christopher¹²,Noller Harry F.¹²

Affiliation:

1. R. Green and H. F. Noller, Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA.

2. C. Switzer, Department of Chemistry, University of California, Riverside, CA 92521, USA.

Abstract

In the ribosome, the aminoacyl–transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23 S ribosomal RNA (rRNA). This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase–catalyzed reaction. This result places the conserved 2555 loop of 23 S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA. Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference31 articles.

1. Unusual Resistance of Peptidyl Transferase to Protein Extraction Procedures

2. Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center.

3. A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome

4. 5′-Dimethoxytrityl- N α -trifluoroacetyl puromycin was attached to alkylamine-derivatized controlled pore glass via a 2′-succinate linkage by standard methods (25). s 4 TCPm was prepared by standard phosphoramidite chemistry with the solid-phase version of puromycin and riboC and 4-thio-dT phosphoramidites (Glen Research). Deprotection was performed by treatment with 3:1 NH 4 OH/MeOH containing 50 mM NaSH overnight at 55°C. Subsequently 40 equivalents of 1 M tetrabutylammonium fluoride in tetrahydrofuran were added to the dried product and incubated overnight at room temperature. Finally 20 mM triethylamine acetate (pH 7.0) was added to the reaction mixture and it was loaded directly on a C 18 high-performance liquid chromatography column and eluted with increasing concentrations of acetonitrile (CH 3 CN). Products were collected and their purity was analyzed on a 20% polyacrylamide 7 M urea gel after 5′ 32 P-end-labeling with polynucleotide kinase.

5. Kinetic characterization of s 4 TCPm was done with the tRNA fragment N -Ac-Met-ACCACC used as a P-site substrate under fragment reaction conditions (18) and with analysis of the resulting product s 4 TCPm- N -Ac-Met by paper electrophoresis. The apparent K m of this compound for the ribosome was 10 μM although at high concentrations peptidyl transferase activity was inhibited (8). This is consistent with binding competition by s 4 TCPm at the P site attributable to wobble pairing between 4-thio-dT (which mimics C74 of tRNA) and G2252 of 23 S rRNA (3); no alternative crosslink associated with P-site occupation by s 4 TCPm was observed.

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