Hsu Lilian M, Han N Natalie
Program in Biochemistry, Mount Holyoke College, South Hadley, Massachusetts, USA.
Program in Biochemistry, Mount Holyoke College, South Hadley, Massachusetts, USA.
J Biol Chem. 2025 Aug 25;301(10):110610. doi: 10.1016/j.jbc.2025.110610.
Productive initiation on the escape rate-limited T5 phage N25 promoter is subject to substantial modulation by the initial transcribed sequence (ITS). It is further compromised by the formation of two classes of open complexes-productive and unproductive. To decipher their roles, we performed single-cycle transcription assays under RNA polymerase (RNAP)-limiting conditions to quantitatively determine the rate of promoter escape and the productive fraction of RNAP open complexes formed at four N25-ITS variant promoters. Each promoter variant was transcribed from three different template conformations, that is, two fragment templates of different lengths and a supercoiled plasmid DNA template. In addition, each time-course transcription reaction was performed in parallel without or with GreB. Our results indicate that ITS variation greatly impacts both parameters, which together determine the extent of productive RNA synthesis from a promoter. Further, both parameters are highly stimulated by template supercoiling, which yields a higher fraction of productive complexes that undergo promoter escape at a faster rate. In contrast, the effect of GreB is selective, showing little effect on RNAP partitioning but increasing the escape rate of N25 variants bearing non-native ITSs. Analysis of the abortive RNA synthesis kinetics on the highly abortive N25anti(-A) promoter reveals the existence of an unproductive ITC making a 7-nt abortive RNA continuously. Based on our new kinetic data and recently published structural information on promoter complexes, we propose for T5 N25 promoters a mechanism of transcription initiation-promoter escape consistent with the roles of the ITS and template supercoiling.
在逃逸速率受限的T5噬菌体N25启动子上的有效起始受到初始转录序列（ITS）的显著调控。两类开放复合物——有效和无效开放复合物的形成进一步削弱了这种调控。为了解析它们的作用，我们在RNA聚合酶（RNAP）受限的条件下进行了单轮转录实验，以定量测定在四个N25-ITS变体启动子处的启动子逃逸速率和形成的RNAP开放复合物的有效部分。每个启动子变体从三种不同的模板构象进行转录，即两种不同长度的片段模板和一个超螺旋质粒DNA模板。此外，每次时程转录反应在无GreB或有GreB的情况下平行进行。我们的结果表明，ITS变异对这两个参数都有很大影响，这两个参数共同决定了启动子进行有效RNA合成的程度。此外，这两个参数都受到模板超螺旋的强烈刺激，超螺旋产生了更高比例的有效复合物，这些复合物以更快的速率进行启动子逃逸。相比之下，GreB的作用具有选择性，对RNAP的分配影响很小，但增加了带有非天然ITS的N25变体的逃逸速率。对高度无效的N25anti(-A)启动子上的流产RNA合成动力学分析揭示了存在一种无效的初始转录复合物，该复合物持续产生一个7个核苷酸的流产RNA。基于我们新的动力学数据和最近发表的关于启动子复合物的结构信息，我们提出了一种与ITS和模板超螺旋的作用相一致的T5 N25启动子转录起始-启动子逃逸机制。
