Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.
Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.
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DOI：
10.1073/pnas.2218516120
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发表时间：
2023-02-14
影响因子：
11.1
通讯作者：
Murakami, Katsuhiko S.
中科院分区：
文献类型：
--
作者：
Vishwakarma, Rishi K.;Qayyum, M. Zuhaib;Babitzke, Paul;Murakami, Katsuhiko S.
RNA聚合酶（RNA polymerase， RNAP）通过转录暂停调控基因表达，控制共转录RNA折叠，使转录与翻译同步，并为调控因子结合提供时间。转录延伸因子NusG通过与转录泡内非模板DNA （ntDNA）链上的保守停顿基序的序列特异性相互作用，刺激革兰氏+细菌（包括枯草芽孢杆菌和结核分枝杆菌）的停顿。我们的结构和生化结果显示，ntDNA中保守的TTNTTT基序的一部分被挤压并夹在NusG和RNAP之间。我们的研究结果进一步表明，RNAP的基本整体构象变化与RNA合成直接相关，NusG-ntDNA相互作用通过干扰这种构象变化来暂停RNA合成。NusG是一种转录延伸因子，通过与转录泡内非模板DNA （ntDNA）链中保守的诱导暂停的- 11TTNTTT - 6基序的序列特异性相互作用，刺激包括枯草芽孢杆菌在内的革兰氏+细菌的转录暂停。为了揭示NusG依赖性暂停的结构基础，我们确定了含有RNA聚合酶（RNAP）、NusG和ntDNA链中TTNTTT基序的暂停转录复合物（PTC）的低温电镜结构。NusG与ntDNA链的相互作用通过在NusG和RNAP的β-叶结构域之间的间隙中定位三个连续的T残基来重新排列转录泡。我们发现，RNAP旋转模块旋转（旋转），使NusG和β叶之间的间隙变宽（旋转状态）和变窄（非旋转状态），是RNAP的内在运动，并与触发环（TL）折叠直接相关，这是所有细胞RNAP在RNA合成反应中必不可少的构象变化。我们还确定了RNAP逃离暂停转录状态的低温电镜结构。这些结构揭示了nusg依赖的暂停机制，通过NusG-ntDNA相互作用抑制从旋转到非旋转状态的转变，从而阻止TL折叠和RNA合成变构。在RNA出口通道内形成的RNA发夹也减少了这种运动。因此，暂停半衰期可以通过NusG-ntDNA相互作用的强度和/或RNA发夹的稳定性来调节。与TTNTTT基序相互作用的NusG残基在细菌中广泛保守，这表明NusG依赖性暂停是普遍存在的。
Transcription pausing by RNA polymerase (RNAP) regulates gene expression where it controls co-transcriptional RNA folding, synchronizes transcription with translation, and provides time for binding of regulatory factors. Transcription elongation factor NusG stimulates pausing in Gram+ bacteria including Bacillus subtilis and Mycobacterium tuberculosis by sequence-specific interaction with a conserved pause motif found in the non-template DNA (ntDNA) strand within the transcription bubble. Our structural and biochemical results revealed that part of the conserved TTNTTT motif in ntDNA is extruded and sandwiched between NusG and RNAP. Our results further demonstrate that an essential global conformational change in RNAP is directly linked to RNA synthesis and that the NusG–ntDNA interaction pauses RNA synthesis by interfering with this conformational change. NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing −11TTNTTT−6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.
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影响因子：
64.5
作者：
Artsimovitch, I;Landick, R
通讯作者：
Landick, R
影响因子：
16
作者：
Gusarov, I;Nudler, E
通讯作者：
Nudler, E
影响因子：
3.2
作者：
Gill, Sumandeep K.;Garcia, George A.
通讯作者：
Garcia, George A.
DOI：
10.1126/science.1251871
发表时间：
2014-05-30
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Larson MH;Mooney RA;Peters JM;Windgassen T;Nayak D;Gross CA;Block SM;Greenleaf WJ;Landick R;Weissman JS
通讯作者：
Weissman JS
影响因子：
4.8
作者：
Crickard, J. Brooks;Fu, Jianhua;Reese, Joseph C.
通讯作者：
Reese, Joseph C.