Single-molecule tracking reveals the functional allocation, in vivo interactions and spatial organization of universal transcription factor NusG
Single-molecule tracking reveals the functional allocation, in vivo interactions and spatial organization of universal transcription factor NusG
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DOI：
10.1101/2022.11.21.517430
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发表时间：
2022-11
期刊：
影响因子：
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通讯作者：
Hafez El Sayyed;Oliver J. Pambos;Mathew Stracy;M. Gottesman;A. Kapanidis
中科院分区：
文献类型：
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作者：
Hafez El Sayyed;Oliver J. Pambos;Mathew Stracy;M. Gottesman;A. Kapanidis
细菌基因表达受到高度调控，以允许细胞生长和适应。许多调控发生在转录延伸过程中，其中 RNA 聚合酶 (RNAP) 在全局且普遍保守的延伸因子 NusG 的帮助下延伸新生 RNA 转录本。 NusG 通过抑制暂停和回溯来调节转录；促进核糖体 RNA (rrn) 操纵子的抗终止； mRNA 基因上的转录与翻译耦合；并刺激有毒基因的 Rho 依赖性终止。尽管对 NusG 进行了大量工作，但其在体内的功能分配和空间分布尚不清楚。在这里，我们利用活大肠杆菌细胞中 NusG 的单分子追踪和超分辨率成像解决了这些长期存在的问题。我们发现，在适度生长的条件下，NusG主要以转录延伸复合物中的RNAP与染色体间接关联的群体存在，并且我们将其鉴定为具有30S核糖体亚基的NusG复合物的缓慢扩散群体；该复合物为 NusG 进入转录延伸提供了一条“30S 引导”的路径。总 NusG 中只有约 10% 是快速扩散的，该群体的流动性表明游离 NusG 在超过 50% 的时间内与 DNA 发生非特异性相互作用。使用抗生素和缺失突变体，我们发现大多数染色体相关的 NusG 参与 rrn 抗终止和转录翻译偶联。 NusG 参与 rrn 抗终止是通过其参与相分离转录凝聚物介导的。我们的工作阐明了中央调节器的各种活动，同时提供了如何使用体内成像剖析多功能机器的作用的指南。
Bacterial gene expression is highly regulated to allow cells to grow and adapt. Much regulation occurs during transcription elongation, where RNA polymerase (RNAP) extends nascent RNA transcripts aided by global and universally-conserved elongation factor NusG. NusG modulates transcription by inhibiting pausing and backtracking; promoting anti-termination on ribosomal RNA (rrn) operons; coupling transcription with translation on mRNA genes; and stimulating Rho-dependent termination on toxic genes. Despite extensive work on NusG, its functional allocation and spatial distribution in vivo is unknown. Here, we addressed these long-standing questions using single-molecule tracking and super-resolution imaging of NusG in live E. coli cells. We found that, under conditions of moderate growth, NusG is mainly present as a population that associates indirectly with the chromosome via RNAP in transcription elongation complexes, and a slowly diffusing population we identified as a NusG complex with the 30S ribosomal subunit; this complex offers a “30S-guided” path for NusG to enter transcription elongation. Only ~10% of total NusG was fast-diffusing, with the mobility of this population suggesting that free NusG interacts non-specifically with DNA for >50% of the time. Using antibiotics and deletion mutants, we showed that most chromosome-associated NusG is involved in rrn anti-termination and in transcriptiontranslation coupling. NusG involvement in rrn anti-termination was mediated via its participation in phase-separated transcriptional condensates. Our work illuminates the diverse activities of a central regulator while offering a guide on how to dissect the roles of multi-functional machines using in vivo imaging.