E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.
E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.
复制标题
大肠杆菌 RNA 聚合酶的 α 亚基 C 端部分被删除，与 lacP1 和 galP1 启动子处的 cAMP-CRP 复合物的相互作用不同。
作者：
A. Kolb;Kazuhiko Igarashi;A. Ishihama;M. Lavigne;M. Buckle;H. Buc
已知RNA聚合酶α-亚基的C-末端部分的缺失根据启动子处CRP结合位点的位置不同地影响由CRP激活的启动子。当CRP结合位点位于-61.5时，如lacP1（I型启动子），激活受到强烈损害，而在galP1（CRP结合信使起始上游41.5 bp处）（II型启动子），激活不受显著影响。我们已经研究了相应的开放复合物的架构中的差异，通过比较定位的全酶重建分别与天然或截短的α-亚基（含有前235或256个残基的）在两个'向上'启动子突变体的lacP1和galP1启动子（分别lacUV5和gal9A16C）。首先，野生型RNA聚合酶对两种启动子的亲和力因CRP和cAMP的存在而增加。相比之下，全酶重建与截短的α-亚基，显示合作的galP1启动子的结合。第二，足迹数据证实了这些观察结果，并表明截短的全酶不能识别启动子上游位置-40的区域。在lacP1启动子处截短的酶和CRP之间不存在接触可以解释激活的缺陷。在galP 1启动子处，其中CRP位点更接近起始位点，截短的聚合酶仍然可以发生蛋白质-蛋白质接触，这表明α亚基的C末端部分不参与激活。
A deletion of the C-terminal part of the alpha-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at -61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at galP1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter). We have investigated the differences in the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated alpha-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and galP1 promoters (respectively lacUV5 and gal9A16C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated alpha-subunits, show cooperative binding at the galP1 promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position -40. The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation. At the galP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the alpha-subunit is not involved in activation.