Said Nelly, Finazzo Mark, Hilal Tarek, Wang Bing, Selinger Tim Luca, Gjorgjevikj Daniela, Artsimovitch Irina, Wahl Markus C
Freie Universität Berlin, Institute of Chemistry and Biochemistry, Laboratory of Structural Biochemistry, Takustr. 6, D-14195 Berlin, Germany.
The Ohio State University, Department of Microbiology and Center for RNA Biology, Columbus, OH, USA.
bioRxiv. 2023 Aug 31:2023.08.30.555460. doi: 10.1101/2023.08.30.555460.
Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits ρ-dependent termination but recapitulation of this activity has proven difficult and the precise mode of Rof action is presently unknown. Our electron microscopic structures of ρ-Rof and ρ-RNA complexes show that Rof undergoes pronounced conformational changes to bind ρ at the protomer interfaces, undercutting ρ conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between ρ and RNA polymerase. Consistently, Rof impedes ρ ring closure, ρ-RNA interactions, and ρ association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirmed that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination . Bioinformatic analyses revealed that Rof is restricted to Pseudomonadota and that the ρ-Rof interface is conserved. Genomic contexts of differ between and , suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence ρ under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by ρ would be lethal.
转录终止因子ρ是一种六聚体的、依赖RNA的NTPase，可呈现活性闭环构象和无活性开环构象。类Sm蛋白Rof是RNA伴侣蛋白Hfq的同源物，它能抑制ρ依赖性终止，但重现这种活性已被证明很困难，目前Rof的精确作用模式尚不清楚。我们对ρ-Rof和ρ-RNA复合物的电子显微镜结构显示，Rof发生显著的构象变化，在原体界面处结合ρ，削弱与环闭合相关的ρ构象动力学，并封闭延伸的初级RNA结合位点，这些位点也是ρ与RNA聚合酶之间界面的一部分。一致地，Rof阻碍ρ环闭合、ρ-RNA相互作用以及ρ与转录延伸复合物的结合。结构导向诱变结合功能分析证实，观察到的ρ-Rof界面是Rof介导的细胞生长抑制和ρ终止所必需的。生物信息学分析表明，Rof仅限于假单胞菌门，并且ρ-Rof界面是保守的。Rof在不同物种间的基因组背景有所不同，这表明Rof的调控模式不同。我们推测，当ρ不受限制的转录终止具有致死性时，Rof和其他细胞抗终止因子在各种尚未确定的应激条件下使ρ沉默。
