Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP
Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP
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DOI：
10.1006/jmbi.2000.4328
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发表时间：
2001-01-26
影响因子：
5.6
通讯作者：
Gourse, RL
中科院分区：
文献类型：
--
作者：
Barker, MM;Gaal, T;Gourse, RL
含有ppGpp的菌株的rRNA启动子活性略低于Delta rel Delta Spot菌株，而氨基酸生物合成/转运启动子活性显著高于Delta rel Delta Spot菌株。ppGpp是一种合成依赖于Eschericlzia COIL的RelA和Spot蛋白的核苷酸。在所附的论文中，我们证明了ppGpp通过缩短a rrn P1开放复合体的寿命在体外直接抑制rRNA启动子的活性。然而，ppGpp在体外不能刺激氨基酸启动子的活性。我们在这里展示了RNA聚合酶(RNAP)突变体，被选来赋予al Delta Rel Delta斑点菌株原生营养，模仿了ppGpp对野生型RNAP的影响。根据突变残基在核心RNAP结构中的位置，我们提出了突变体如何降低开放复合体寿命的分子模型，类比ppGpp。我们发现，氨基酸启动子在体外和体内需要比对照启动子更高浓度的RNAP来发挥作用，并且在体内对RNAP的竞争比对照启动子更敏感。此外，我们还表明，通过增加ppGpp的限速RNAP结合步骤，可以减轻体内对ppGpp的氨基酸启动子的需求。我们的数据与之前提出的被动模型一致，在该模型中，ppGpp通过缩短rrn P1开放复合体的寿命，释放足够的RNAP来刺激氨基酸启动子的转录，从而直接抑制稳定的RNA合成。我们的数据还对引用可能以ppGpp依赖的方式增加氨基酸启动子活性的假设因素的模型施加了相当大的限制。(C)2001年学术出版社。
Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Eschericlzia coil, display slightly lower rRNA promoter activity and much higher amino acid biosynthesis/transport promoter activity than Delta rel Delta spoT strains. In the accompanying paper, we show that ppGpp directly inhibits rRNA promoter activity in vitro by decreasing the lifetime of the a rrn P1 open complex. However, ppGpp does not stimulate amino acid promoter activity in vitro. We show here that RNA polymerase (RNAP) mutants, selected to confer prototrophy to al Delta rel Delta spoT strains, mimic the effects of ppGpp on wild-type RNAP. Based on the positions of the mutant residues that confer prototrophy in the structure of core RNAP, we suggest molecular models for how the mutants, and by analogy ppGpp, generally decrease the lifetime of open complexes. We show that amino acid promoters require higher concentrations of RNAP for function in vitro and in vivo than control promoters, and are more sensitive to competition for RNAP in vivo than control promoters. Furthermore, we show that the requirement of an amino acid promoter for ppGpp in vivo can be alleviated by increasing its rate-limiting RNAP-binding step. Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthesis directly by reducing the Lifetime of the rrn P1 open complex, Liberating enough RNAP to stimulate transcription from amino acid promoters. Our data also place considerable constraints on models invoking hypothetical factors that might increase amino acid promoter activity in a ppGpp-dependent fashion. (C) 2001 Academic Press.