High-Resolution Structure/Function Analysis of the Strand-Loop Network of RNA Polymerase

The majority of cells contain the same genetic material that encodes the structure of various cellular components. Differences in cell types (e.g. neurons as compared to muscle cells) arise because different parts of the genetic material are selectively read out. RNA polymerases (RNAPs) are a crucial part of the molecular machinery involved in this selective reading mechanism. To understand normal and pathological cellular conditions better we need to gain an improved insight of the molecular mechanisms underlying RNAP function. Such knowledge will result in more specifically targeted therapeutic applications, new diagnostic tools and help with the development of novel types of antibiotica

The proposal describes an experimental strategy aimed at the functional characterization of several key functional domains required for the catalytic function of RNA polymerase (RNAP). My laboratory developed a unique model system based on the in vitro assembly of a functionally active archaeal RNAP from recombinant subunits. Archaeal RNAP are very similar in structure and function to eukaryotic RNAPII. We can therefore take maximal advantage of the high-resolution structures available for various bacterial RNAPs and yeast RNAPII to create a series of targeted mutations in several structural domains (such as the lid, rudder and fork loop 1) that play a key role in the catalytic and translocation functions by interacting with the nucleic acid substrates during the transcription process. We will use an efficient mutagenesis strategy based on silently mutated epxression vectors containing strategically places restriction eznyme target sites to insert doubles-stranded oligonucleotides carrying the desired mutations directly into bacterial expression vectors. This will allow us to efficiently produce a large (saturating) number of mutants. The effects of these changes will be studied using a variety of recruitment, transcription, translocation and elongation assays with the aim of identifying the positions and functional roles of key residues within these domains. I anticipate that many of the insights obtained in this system will be directly applicable for enhancing our understanding of some of the most fundamental aspects of eukaryotic transcription mechanisms.