Understanding gene expression in bacteria has produced health benefits ranging from biotechnology to cancer therapy. The molecular mechanism of gene transcription can be studied with high resolution by techniques that share a common basis in chemistry. Previously, we have designed experiments that identify the macromolecules contacted by the leading end of nascent RNA at almost every step of its path through the transcription complex which catalyzes its synthesis. The next task is to understand the number and nature of the binding sites that form the RNA path. We propose to apply new methods to identify the individual residues in the proteins and DNA contacted by nascent RNA as it passes through the transcription complex, providing a wealth of information about the process. 1. Using an artificial protease---a small metal chelate that can be activated when desired, which we conceived and developed during the current period---we shall map those amino-acid residues in the enzyme subunits that come into contact with the leading end of the nascent RNA molecule. This will be done by preparing complexes containing defined lengths of RNA without protease at the leading end, and then activating the protease. Our protease works by proximity, not by residue type; it selectively cleaves peptide bonds directly adjacent to it. Isolation of the new fragments and sequencing around the cleavage sites will provide a high resolution map of those amino acid residues forming the path of RNA across the enzyme's surface, along with their positions in the primary structures of the enzyme subunits involved. Recent advances in the technology of preparing stable transcription complexes containing defined lengths of RNA have significantly altered our experimental protocols. We shall investigate and compare transcription complexes made (i) using dinucleotide initiators and selected chain terminators and (2) using artificial transcription bubbles made from machine-synthesized nucleic acids. 2. We shall determine the RNA lengths and DNA sites at which the leading end of the transcript contacts the DNA template in transcription complexes. Several promoters will be compared, since the DNA sequence is likely to be an important factor. This will be carried out (1) using an aromatic azide at the leading end of a dinucleotide primer, producing a collection of terminated transcription complexes, photolyzing it and isolating and characterizing the photo-crosslinked RNA/DNA conjugate; and (2) using limited elongation to prepare a set of artificial transcription bubbles each with a probe attached to the leading end of RNA.
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