Use of Site-Specific Protein-DNA Photocrosslinking to Analyze the Molecular Organization of the RNA Polymerase II Initiation Complex

Abstract

Site-specific protein-DNA photocrosslinking has proved to be the method of choice for analysis of the formation of nucleoprotein complexes such as those involved in transcription by mammalian RNA polymerase II (RNA Pol II). The method has two principal advantages. First, it yields structural information on large, multisubunit complexes that in general cannot be analyzed using standard high-resolution techniques such as X-ray crystallography or nuclear magnetic resonance (NMR). For example, site-specific protein-DNA photocrosslinking, in conjunction with complementary methods such as protein-affinity chromatography and electron microscopy, has produced information on both the molecular organization and the composition of the RNA Pol II pre-initiation complex on promoter DNA (1–4). This complex contains RNA Pol II and the general transcription factors TBP, TFIIA, TFIIB, TFIIE, TFIIF (RAP74 and RAP30), and TFIIH, and is composed of more than 25 polypep-tides ranging in Mr from 10 to 220 kDa (5). Neither X-ray crystallography nor NMR, which can only resolve the structure of complexes containing short protein fragments bound to small pieces of promoter DNA, could provide any detailed structural information on this complex. Second, the method has sufficient technical flexibility so as to allow the rapid analysis of complexes assembled under various conditions. Over the past few years, we have analyzed a large collection of complexes assembled in the presence of various combinations of the general transcription factors (wild-type or different deletion mutants) and RNA Pol II (1–4). These experiments have enabled us to draw conclusions on the dynamics of RNA Pol II pre-initiation complex assembly and has led to the notion that isomerization of the RNA Pol II pre-initiation complex proceeds through wrapping of the promoter DNA around the enzyme (4).