Schroeder Lisa A, Karpen Mary E, deHaseth Pieter L
Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
J Mol Biol. 2008 Feb 8;376(1):153-65. doi: 10.1016/j.jmb.2007.11.070. Epub 2007 Nov 28.
Initiation of transcription is an important target for regulation of gene expression. In bacteria, the formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of Escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for threonine (T) at position 429 of sigma(70): its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by T429A substitution, thus providing evidence for a role of T429 in the strand-separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding that stabilizes the highly conserved -11A-T base pairs of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the -10 region. This model suggests an active role for RNA polymerase in disrupting the -11 base pair, rather than just capturing the -11A subsequent to spontaneous unpairing.
转录起始是基因表达调控的一个重要靶点。在细菌中，RNA聚合酶与启动子之间形成转录活性复合物涉及一段约14个碱基对的DNA链分离。已发现大肠杆菌σ⁷⁰保守区域2.3中的芳香族和碱性残基参与这一过程，但仍不清楚是哪些氨基酸残基启动了这一过程。在此，我们报道了σ⁷⁰第429位苏氨酸（T）的关键作用：用丙氨酸取代它（T429A）导致开放复合物形成的减少幅度是区域2.3中任何单个取代所观察到的最大降幅。启动子识别本身不受T429A取代的影响，从而为T429在链分离步骤中的作用提供了证据。我们的数据与一个模型一致，即T429会作为氢键的竞争者，氢键稳定启动子DNA高度保守的-11A-T碱基对，从而促进在-10区域这个特定位置的链分离起始。该模型表明RNA聚合酶在破坏-11碱基对中发挥积极作用，而不仅仅是在自发解链后捕获-11A。
