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Transcription factors modulate RNA polymerase conformational equilibrium.

ABSTRACT: RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this role. Their structural effects are consistent with biochemical results on two categories of transcriptional pauses. In addition, the structures suggest a cooperative mechanism of NusA and NusG during Rho-mediated transcription termination. Our results provide a structural rationale for the stochastic nature of pausing and termination and how NusA and NusG can modulate it.

SUBMITTER: Zhu C

PROVIDER: S-EPMC8940904 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Transcription factors modulate RNA polymerase conformational equilibrium.

Zhu Chengjin C Guo Xieyang X Dumas Philippe P Takacs Maria M Abdelkareem Mo'men M Vanden Broeck Arnaud A Saint-André Charlotte C Papai Gabor G Crucifix Corinne C Ortiz Julio J Weixlbaumer Albert A

Nature communications 20220322 1

RNA polymerase (RNAP) frequently pauses during the transcription of DNA to RNA to regulate gene expression. Transcription factors NusA and NusG modulate pausing, have opposing roles, but can bind RNAP simultaneously. Here we report cryo-EM reconstructions of Escherichia coli RNAP bound to NusG, or NusA, or both. RNAP conformational changes, referred to as swivelling, correlate with transcriptional pausing. NusA facilitates RNAP swivelling to further increase pausing, while NusG counteracts this ...[more]

PMID: 35318334

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Project description:Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

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Transcription factors modulate RNA polymerase conformational equilibrium.

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Transcription factors modulate RNA polymerase conformational equilibrium.

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