Chapter 23

Applying Photolabile Derivatives of Oligonucleotides To Probe the Peptidyltransferase Center

Barry S. Cooperman

Barry S. Cooperman

Department of Chemistry, University of Pennsylvania, Philadelphia, PA, 19104-6323

Search for more papers by this author
Serguei N. Vladimirov

Serguei N. Vladimirov

Department of Chemistry, University of Pennsylvania, Philadelphia, PA, 19104-6323

Search for more papers by this author
Yuri Bukhtiyarov

Yuri Bukhtiyarov

DuPont Pharmaceuticals Co., Experimental Station E400/3410, Wilmington, DE, 19880

Search for more papers by this author
Zhanna Druzina

Zhanna Druzina

Department of Chemistry, University of Pennsylvania, Philadelphia, PA, 19104-6323

Search for more papers by this author
Ruo Wang

Ruo Wang

Schering-Plough Research Institute, 2015 Galloping Hill Rd., K-15-3-3545, Kenilworth, NJ, 07033-0539

Search for more papers by this author
Hyuk-Soo Seo

Hyuk-Soo Seo

Department of Chemistry, University of Pennsylvania, Philadelphia, PA, 19104-6323

Search for more papers by this author
First published: 27 March 2000

Summary

The chapter describes an approach to form defined photo-cross-links from targeted RNA sites within the ribosome, with an emphasis on those that are important functionally. In this approach, radioactive, photolabile derivatives of oligonucleotides (PHONTs) having sequences complementary to rRNA sequences are bound to their targeted sequences in intact ribosomal subunits and, on photolysis, form cross-links with neighboring ribosomal components. The PHONT approach offers several advantages. First, it allows targeting of sequences of particular functional or structural significance throughout the ribosome structure. Second, the cross-links formed provide a defined upper-limit distance for the separation of the linked components within the ribosome, given by the length of the tether. The chapter first describes the PHONT approach in general before presenting the results of recent applications of the approach to the study of the peptidyltransferase center (PTC). It identifies four principal elements in PHONT design: first, the backbone structure; second, the placement of the photolabile group within the oligonucleotide sequence; third, the length and flexibility of the tether linking the photolabile group with the oligonucleotide backbone; and fourth, the introduction of radioactivity. As is evident in the descriptions, PHONT design continues to evolve. Currently the YAMMP approach is applied to develop a three-dimensional model of the PTC based on cross-linking results, which provide the clearest set of constraints for model construction.

The full text of this article hosted at iucr.org is unavailable due to technical difficulties.