Dissecting the functions of mycobacteria RbpA structural domains

RNA polymerase (RNAP) binding protein RbpA contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Based on structural and biochemical data, four domains have been identified in the RbpA protein: a N-terminal tail (NTT) domain of unknown function, a core domain (CD) that contacts the RNAP ß' subunit in a recently solved crystal structure, a basic linker (BL) that binds DNA, and a s-interaction domain (SID) that binds group I and group II s-factors. However, limited in vivo studies have been performed in mycobacteria and how the individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We dissected the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis. We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP holoenzyme, indicating that the primary function of the CD is not solely association with the RNAP. We show that RbpA BL and SID are required for stabilization of RPo complexes at the ribosomal RNA rrnAP3 promoter in vitro, while the NTT and CD are dispensable. Finally, we determine that the NTT and CD impact gene expression of a distinct set of genes from that affected by the BL and SID activities. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. Overall design: Transcriptomes of 4 Mycobacterium smegamtis strains including csm275 (WT RbpA), csm314 (RbpA R88A), csm322 (RbpA R79A) and csm328 (RbpA delta 1-71), which express different RbpA alleles are compared in triplicate.

RNA聚合酶（RNA polymerase, RNAP）结合蛋白RbpA可促进稳定的RNA聚合酶-启动子开放复合物（RPo）的形成，且对分枝杆菌的存活至关重要。基于结构与生物化学数据，研究人员已在RbpA蛋白中鉴定出四个结构域：功能未知的N端尾部（N-terminal tail, NTT）结构域、在近期解析的晶体结构中可与RNA聚合酶β'亚基结合的核心结构域（core domain, CD）、结合DNA的碱性接头（basic linker, BL），以及可结合I组与II组σ因子的σ相互作用结构域（σ-interaction domain, SID）。然而，目前在分枝杆菌中开展的体内研究较为有限，RbpA各独立结构域如何参与RbpA功能发挥及分枝杆菌基因表达调控，目前仍不甚明确。我们利用一系列靶向RbpA各独立结构域的rbpA基因突变体，剖析了分枝杆菌中RbpA各结构域的功能作用。各RbpA结构域的功能对于结核分枝杆菌（Mycobacterium tuberculosis）的存活以及耻垢分枝杆菌（Mycobacterium smegmatis）的最佳生长均不可或缺。我们发现，RbpA的σ相互作用结构域（SID）既必需又足以介导RbpA与RNA聚合酶全酶的相互作用，这表明核心结构域（CD）的主要功能并非仅为与RNA聚合酶结合。我们的实验显示，在体外条件下，RbpA的碱性接头（BL）与σ相互作用结构域（SID）是稳定核糖体RNA rrnAP3启动子处RPo复合物所必需的，而N端尾部（NTT）与核心结构域（CD）则非必需。最后，我们确认N端尾部（NTT）与核心结构域（CD）所调控的基因表达谱，与碱性接头（BL）及σ相互作用结构域（SID）活性所调控的基因集存在显著差异。本研究结果阐明了RbpA各独立功能结构域发挥活性的具体效应。实验整体设计：选取4株表达不同RbpA等位基因的耻垢分枝杆菌（Mycobacterium smegmatis）菌株，分别为csm275（野生型RbpA）、csm314（RbpA R88A突变体）、csm322（RbpA R79A突变体）与csm328（RbpA Δ1-71突变体），对其转录组进行三次生物学重复比较分析。