Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
Nat Struct Mol Biol. 2010 Jan;17(1):99-104. doi: 10.1038/nsmb.1732. Epub 2009 Dec 6.
The trigger loop (TL) is a polymorphous component of RNA polymerase (RNAP) that makes direct substrate contacts and promotes nucleotide addition when folded into an alpha-helical hairpin (trigger helices, TH). However, the roles of the TL/TH in transcript cleavage, catalysis, substrate selectivity and pausing remain ill defined. Based on in vitro assays of Escherichia coli RNAP bearing specific TL/TH alterations, we report that neither intrinsic nor regulator-assisted transcript cleavage of backtracked RNA requires formation of the TH. We find that the principal contribution of TH formation to rapid nucleotidyl transfer is steric alignment of the reactants rather than acid-base catalysis, and that the TL/TH cannot be the sole contributor to substrate selectivity. The similar effects of TL/TH substitutions on pausing and nucleotide addition provide additional support for the view that TH formation is rate-limiting for escape from nonbacktracked pauses.
触发环（TL）是 RNA 聚合酶（RNAP）的一种多态性组成部分，当折叠成α-螺旋发夹（触发螺旋，TH）时，它与底物直接接触并促进核苷酸的添加。然而，TL/TH 在转录切割、催化、底物选择性和暂停中的作用仍未得到明确界定。基于对具有特定 TL/TH 改变的大肠杆菌 RNAP 的体外测定，我们报告说，无论是内在的还是调节剂辅助的回溯 RNA 的转录切割都不需要 TH 的形成。我们发现，TH 形成对快速核苷酸转移的主要贡献是反应物的空间排列而不是酸碱催化，并且 TL/TH 不可能是底物选择性的唯一贡献者。TL/TH 取代对暂停和核苷酸添加的相似影响为 TH 形成是从非回溯暂停中逃逸的限速步骤的观点提供了额外的支持。
