The molecular mechanism of gene transcription can be studied with high resolution by techniques that share a common basis in chemistry. Our overall goal for the next five years is to determine where the individual enzyme subunits bind to core RNA polymerase, DNA, and RNA in transcription complexes. Primarily this involves preparing a panel of mutants of a protein such as the E. coli alpha or sigma subunit, with each mutant containing a single reactive site for attachment of cutting reagent. This reagent, FeBABE, works by proximity, not by residue type; it selectively cleaves nearby peptide or nucleic acid backbones. Each protein conjugate will be used as a probe to cut other macromolecules in a specific complex. We shall map those residues in proteins or nucleic acids that are next to the tethered reagent at various stages of the transcription process: core enzyme, holoenzyme, binary enzyme-DNA complex, ternary enzyme-DNA-enhancer complex, ternary enzyme-DNA-RNA complex, etc. The information gained from this panel of mutants will be supplemented by other strategies, such as conjugating each with a panel of homologous cutting reagents able to cleave macromolecules over an adjustable range of distances. Taken to their logical limit, these experiments will provide a surface map of the entire complex. Developing the methodology for high resolution study of these megadalton complexes will produce tools that may find widespread use in surveying the architecture of other large molecular assemblies. These reagents are powerful probes of macromolecular structure, usable with relative ease in any system where tethered reagents are applicable. They are well suited to the study of proximity relationships in multi-subunit protein-nucleic acid complexes. The cleavage reaction is fast (seconds) , selective, and proceeds in high yield under physiological conditions. It forms products that can be characterized by standard N-terminal sequencing of the resolved peptides or by gel electrophoresis. Cleavage of proteins and nucleic acids occurs under the same conditions, permitting study of all the macromolecules involved in transcription complexes. Basic understanding of gene expression in bacteria has already formed the foundation of biotechnology; more detailed knowledge will almost certainly produce new therapeutic targets for the next generation of antibiotics.
Showing the most recent 10 out of 30 publications
