Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Nucleic Acids Res. 2024 May 8;52(8):4556-4574. doi: 10.1093/nar/gkae209.
Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). A surface-exposed domain inserted into the catalytic trigger loop (TL) of Escherichia coli RNAP, called SI3, modulates pausing and is essential for growth. Here we describe a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution (β'Ala941→Thr; ΔSI3*). ΔSI3* increased transcription rate in vitro relative to ΔSI3, possibly explaining its viability, but retained both positive and negative effects of ΔSI3 on pausing. ΔSI3* inhibited pauses stabilized by nascent RNA structures (pause hairpins; PHs) but enhanced other pauses. Using NET-seq, we found that ΔSI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at ΔSI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. ΔSI3*-suppressed pauses also were associated with apparent pausing one nucleotide upstream from the consensus sequence, often generating tandem pause sites. These '-2 pauses' were stimulated by pyrophosphate in vitro and by addition of apyrase to degrade residual NTPs during NET-seq sample processing. We propose that some pauses are readily reversible by pyrophosphorolysis or single-nucleotide cleavage. Our results document multiple ways that SI3 modulates pausing in vivo and may explain discrepancies in consensus pause sequences in some NET-seq studies.
转录暂停通过细胞 RNA 聚合酶（RNAP）辅助基因调控。大肠杆菌 RNAP 催化触发环（TL）中插入的一个表面暴露结构域，称为 SI3，调节暂停，对生长至关重要。本文描述了一种可行的大肠杆菌菌株，该菌株缺乏 SI3，但通过 TL 取代（β'Ala941→Thr；ΔSI3*）得以维持。与 ΔSI3 相比，ΔSI3在体外增加了转录速率，这可能解释了其生存能力，但保留了 ΔSI3 对暂停的正反两方面影响。ΔSI3抑制了由新生 RNA 结构（暂停发夹；PHs）稳定的暂停，但增强了其他暂停。使用 NET-seq，我们发现 ΔSI3*-增强的暂停类似于共识基本暂停序列，而 ΔSI3*-抑制的暂停序列与 PHs 更相关，序列更为多样化。ΔSI3*-抑制的暂停也与在共识序列上游一个核苷酸处的明显暂停相关，通常会产生串联暂停位点。这些 '-2 暂停'在体外受到焦磷酸的刺激，并通过添加核酸外切酶在 NET-seq 样品处理过程中降解残留的 NTP。我们提出，一些暂停很容易通过焦磷酸解或单核苷酸切割来逆转。我们的结果记录了 SI3 在体内调节暂停的多种方式，这可能解释了一些 NET-seq 研究中共识暂停序列的差异。
