Probing the Organization of Transcription Complexes Using Photoreactive 4-Thio-Substituted Analogs of Uracil and Thymidine

This chapter analyzes the probing of the organization of transcription complexes using photoreactive 4-Thio-substituted analogs of uracil and thymidine. The use of 4-thio-substituted UTP or dTTP analogs provides a powerful method to probe the organization of transcription complexes. These analogs may be placed at defined positions in the transcript or the DNA, and on UV activation form cross-links to nearby regions of the protein with high efficiency. A number of analogs have been employed in these studies. Subsequent mapping of the site of the cross-link by peptide mapping provides important information concerning the organization of the complex and the trajectory of the nucleic acid components over the surface of the protein. The preparation of transcription complexes with specifically positioned photoreactive analogs is experimentally designed. The design of the template is dictated by the purpose of the experiment and the position in the template or product that is being analyzed. The exact strategy for mapping the site of the cross-link depends on the suitability of various chemical or enzymatic cleavage reagents with the particular protein being examined.