Chadsey M S, Karlinsey J E, Hughes K T
Department of Microbiology, University of Washington, Seattle, Washington 98195, USA.
Genes Dev. 1998 Oct 1;12(19):3123-36. doi: 10.1101/gad.12.19.3123.
The anti-sigma factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific sigma factor, sigma28. FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free sigma28. We have analyzed FlgM-mediated inhibition of sigma28 activity in vitro. FlgM is able to inhibit sigma28 activity even when sigma28 is first allowed to associate with core RNAP. Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both sigma28 and sigma28 holoenzyme (Esigma28). The Kd of the sigma28-FlgM complex is approximately 2 x 10(-10) M; missense mutations in FlgM that cause a defect in sigma28 inhibition in vivo increase the Kd of this interaction by 4- to 10-fold. SPR measurements of Esigma28 dissociation in the presence of FlgM indicate that FlgM destabilizes Esigma28, presumably via an interaction with the sigma subunit. Our data provide the first direct evidence of an interaction between FlgM and Esigma28. We propose that this secondary activity of FlgM, which we term holoenzyme destabilization, enhances the sensitivity of the cell to changes in FlgM levels during flagellar biogenesis.
鼠伤寒沙门氏菌的抗σ因子FlgM通过与鞭毛特异性σ因子σ28直接相互作用，抑制3类鞭毛基因的转录。据信，FlgM通过隔离游离的σ28来阻止RNA聚合酶（RNAP）全酶的形成。我们在体外分析了FlgM介导的对σ28活性的抑制作用。即使首先让σ28与核心RNAP结合，FlgM也能够抑制σ28的活性。表面等离子体共振（SPR）用于评估FlgM与σ28以及σ28全酶（Eσ28）之间的相互作用。σ28-FlgM复合物的解离常数（Kd）约为2×10^(-10) M；在体内导致σ28抑制缺陷的FlgM错义突变使这种相互作用的Kd增加4至10倍。在存在FlgM时对Eσ28解离的SPR测量表明，FlgM使Eσ28不稳定，推测是通过与σ亚基相互作用。我们的数据首次提供了FlgM与Eσ28之间相互作用的直接证据。我们提出，FlgM的这种二级活性，我们称之为全酶不稳定化，增强了细胞在鞭毛生物合成过程中对FlgM水平变化的敏感性。
