Lehrstuhl Biopolymere, Universität Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany.
Forschungszentrum für Bio-Makromoleküle, Universität Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany.
Nat Commun. 2019 Feb 11;10(1):702. doi: 10.1038/s41467-019-08567-6.
RfaH, member of the NusG/Spt5 family, activates virulence genes in Gram-negative pathogens. RfaH exists in two states, with its C-terminal domain (CTD) folded either as α-helical hairpin or β-barrel. In free RfaH, the α-helical CTD interacts with, and masks the RNA polymerase binding site on, the N-terminal domain, autoinhibiting RfaH and restricting its recruitment to opsDNA sequences. Upon activation, the domains separate and the CTD refolds into the β-barrel, which recruits a ribosome, activating translation. Using NMR spectroscopy, we show that only a complete ops-paused transcription elongation complex activates RfaH, probably via a transient encounter complex, allowing the refolded CTD to bind ribosomal protein S10. We also demonstrate that upon release from the elongation complex, the CTD transforms back into the autoinhibitory α-state, resetting the cycle. Transformation-coupled autoinhibition allows RfaH to achieve high specificity and potent activation of gene expression.
RfaH 是 NusG/Spt5 家族的成员,可激活革兰氏阴性病原体中的毒力基因。RfaH 存在两种状态,其 C 端结构域(CTD)要么折叠成α-螺旋发夹,要么折叠成β-桶。在游离的 RfaH 中,α-螺旋 CTD 与 N 端结构域上的 RNA 聚合酶结合位点相互作用,并掩盖该位点,从而自动抑制 RfaH 并限制其募集到 opsDNA 序列。激活后,两个结构域分离,CTD 重新折叠成β-桶,从而招募核糖体,激活翻译。我们使用 NMR 光谱法表明,只有完整的 ops 暂停转录延伸复合物才能激活 RfaH,可能是通过瞬时接触复合物,允许重新折叠的 CTD 结合核糖体蛋白 S10。我们还证明,在从延伸复合物释放后,CTD 转变回自动抑制的α状态,重置循环。转换偶联的自动抑制使 RfaH 能够实现高特异性和强效的基因表达激活。