Mycobacterial RNA polymerase requires a U-tract at intrinsic terminators and is aided by NusG at suboptimal terminators.
Mycobacterial RNA polymerase requires a U-tract at intrinsic terminators and is aided by NusG at suboptimal terminators.
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DOI：
10.1128/mbio.00931-14
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发表时间：
2014-04-08
期刊：
影响因子：
6.4
通讯作者：
Landick R
中科院分区：
文献类型：
--
作者：
Czyz A;Mooney RA;Iaconi A;Landick R
内源性终止子编码富含GC的RNA发夹，紧随其后的是富含7-9个核苷酸(NT)的U-链，在大多数细菌中起着标点和调节转录的主要作用。然而，在一些细菌谱系中，具有较强U-链的典型内在终止子的表达不足，这导致了他们的RNA聚合酶停留在编码各种缺乏U-链的RNA结构的非规范内在终止子上。我们构建了分枝杆菌RNA聚合酶及其主要延伸因子NusA和NusG的重组形式，以表征分枝杆菌的内在终止性。使用没有可能的分枝杆菌污染的体外转录实验，我们证明了分枝杆菌RNA聚合酶比大肠杆菌RNA聚合酶更有效地终止于具有不完整U-链的典型终止子，但即使在存在分枝杆菌NusA和NusG的情况下，也不能终止于假定的缺乏U-链的终止子。然而，分枝杆菌NusG显示出一种新的终止刺激活性，可能允许具有次优U-链的固有终止子有效地发挥作用。细菌依赖于转录终止来定义和调节基因表达单位。在大多数细菌中，精确的终止和许多衰减的调节是由内在终止子完成的，这些终止子编码富含GC的发夹和U-链，是破坏稳定的转录延伸复合体所必需的。因此，在分枝杆菌中明显缺乏具有可识别U-链的规范固有终止子是非常有意义的，因为非规范固有终止子可以揭示破坏转录复合体稳定的新途径，而且因为对终止的准确理解对于对抗分枝杆菌疾病的策略和一般的计算生物信息学是至关重要的。我们的发现分枝杆菌RNA聚合酶需要U-链进行内在终止，这可以在NusG的帮助下进行，这将指导未来对分枝杆菌转录的研究，并有助于改进预测算法来注释细菌基因组序列。
Intrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich “U-tract,” play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently. Bacteria rely on transcription termination to define and regulate units of gene expression. In most bacteria, precise termination and much regulation by attenuation are accomplished by intrinsic terminators that encode GC-rich hairpins and U-tracts necessary to disrupt stable transcription elongation complexes. Thus, the apparent dearth of canonical intrinsic terminators with recognizable U-tracts in mycobacteria is of significant interest both because noncanonical intrinsic terminators could reveal novel routes to destabilize transcription complexes and because accurate understanding of termination is crucial for strategies to combat mycobacterial diseases and for computational bioinformatics generally. Our finding that mycobacterial RNA polymerase requires U-tracts for intrinsic termination, which can be aided by NusG, will guide future study of mycobacterial transcription and aid improvement of predictive algorithms to annotate bacterial genome sequences.