High-Resolution Mutagenesis of the 'Bridge Helix' and 'Switch 1' Domains of Archaeal RNA Polymerase

The majority of cell types contain the same genetic material to encode the various cellular components. Differences in cell types (e.g. neurons as compared to muscle cells) arise because different parts of the genetic material are read out. RNA polymerases (RNAPs) are a crucial part of the cellular machinery involved in the selective reading mechanism (transcription) and it is therefore important to understand their molecular structure and function in considerable detail. By studying the chemical and physical mechanisms underlying RNAP function we can begin to understand the way these mechanisms are controlled within the cell to reflect the different requirements for gene expression that are posed by various metabolic, developmental and disease states. In this project we intend to study two key structures that are involved directly in the function of RNAP to obtain an insight into the underlying molecular mechanisms.

The proposal aims for a detailed characterization of the functional properties of two key structural domains involved in the catalytic process of RNA polymerase (RNAP). We have developed a unique model system (from the hyperthermophile Methanocaldococcus jannaschii) that allows us to assemble an active RNAP from recombinant subunits. Archaeal RNAPs are structurally and functionally very close to eukaryotic RNAPs, especially RNAPII. In addition, many functional elements of the catalytic site are highly conservered in bacterial RNAP. We can therefore take advantage of the high-resolution structures available for bacterial RNAPs and yeast RNAPII to design a series of targeted mutations in two highly conserved structures of the archaeal enzyme, the bridge helix and switch-1 domain. We will use an efficient mutagenesis strategy based on silently-mutated expression vectors (with unique restriction enzyme sites) to insert double-stranded oligonucleotides carrying the desired changes directly into bacterial expression vectors. This will allow us to generate a large number of mutants designed to obtain further insights into the catalytic mechanisms used during transcription intitiation and elongation. We anticipate that many of the findings from archaeal enzymes will be directly applicable for an understanding of eukaryotic transcription mechanisms.