CHEMICAL PROBES OF RNA POLYMERASE AND DNA

了解细菌中的基因表达对健康有益 从生物技术到癌症治疗。的分子机制 基因转录可以用高分辨率的技术来研究， 在化学上有共同的基础。在此之前，我们设计了 鉴定前端接触的大分子的实验 新生RNA在转录过程中的几乎每一步 催化其合成的络合物。下一个任务是了解 形成RNA路径的结合位点的数量和性质。我们建议 应用新的方法来鉴定蛋白质中的单个残基， 而新生RNA在转录过程中与DNA接触 复杂，提供了有关该过程的丰富信息。 1.使用人工蛋白酶-一种小的金属螯合物， 当我们想要的时候，我们就会被激活， 我们将绘制酶中的氨基酸残基 与新生RNA的前端接触的亚基 分子。这将通过制备含有定义的 在前端没有蛋白酶的RNA长度，然后激活 蛋白酶我们的蛋白酶通过邻近而不是残基类型起作用;它 选择性地切割与其直接相邻的肽键。 新的片段和切割位点周围的测序将提供一个 形成RNA路径的氨基酸残基的高分辨率图谱 沿着它们在初级酶中的位置， 所涉及的酶亚基的结构。的最新进展 一种制备稳定的含有特定结构的转录复合物的技术 RNA的长度已经显著改变了我们的实验方案。我们 将调查和比较转录复合物（i）使用 二核苷酸引发剂和选择的链终止剂，和（2）使用 由机器合成的核酸制成的人工转录泡 acids. 2.我们将确定RNA长度和DNA位点， 转录物的末端在转录中接触DNA模板 配合物将比较几种启动子，因为DNA序列是 可能是一个重要因素。这将通过（1）使用 在二核苷酸引物的前端处的芳香族叠氮化物，产生 收集终止的转录复合物，将其光解， 分离和表征光交联的RNA/DNA缀合物;和 (2)使用有限的延伸来制备一组人工转录， 每个气泡都有一个探针连接到RNA的前端。

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.