Bacteriophage T4 Gene Expression

正常的细胞发育需要调控转录起始和激活，以便在适当的时间表达适当的基因。我们使用一个简单的原核系统研究转录起始的调控：大肠杆菌RNA聚合酶，一个由核心(2 α, β, β ？，和)和sigma特异性因子。sigma70因子指定在营养生长过程中负责基础基因表达的启动子的转录。当sigma70存在于聚合酶中时，它的两个结构域，2.4（内部区域）和4.2 （c端区域），与？10和？宿主启动子DNA的35个区域。我们小组的一个重点是了解RNA聚合酶的成分如何影响启动子的利用。RNA聚合酶的sigma70亚基只有在与核心RNA聚合酶结合并形成全酶后才能与启动子DNA相互作用。游离的sigma70被sigma70的n端100个氨基酸（结构域1.1）所抑制，从而阻断了结构域4.2与DNA的相互作用。此外，在free sigma70中，域2.4和4.2离得太近了。10和？35个启动子区域。sigma70与核心结合后发生构象变化，揭开结构域4.2，增加结构域2.4和4.2之间的间距。许多实验室先前的工作表明，sigma结构域2.4和4.2与各自的识别元件的相互作用有相当严格的要求，分别距离转录开始10和35个碱基对。

Normal cell development requires regulation of transcription initiation and activation in order to express appropriate genes at appropriate times. We study regulation of transcription initiation using a simple prokaryotic system: E. coli RNA polymerase, a five subunit complex comprised of a core (2 alphas, beta, beta?, and omega) and a sigma specificity factor. The sigma70 factor specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma70 is present within polymerase, two of its domains, 2.4 (an internal region) and 4.2 (the C-terminal region), interact with sequences within the ?10 and ?35 regions, respectively, of host promoter DNA. One focus of our group has been to understand how the components of RNA polymerase affect promoter utilization. The sigma70 subunit of RNA polymerase interacts with promoter DNA only after binding to core RNA polymerase and forming the holoenzyme. Free sigma70 is inhibited from promoter binding by the N-terminal 100 amino acids of sigma70 (domain 1.1), which block domain 4.2 interaction with DNA. Additionally, in free sigma70, domains 2.4 and 4.2 are too close together for the distance between the ?10 and ?35 promoter regions. Upon binding to core, sigma70 undergoes a conformational change, which unmasks domain 4.2 and increases the spacing between domains 2.4 and 4.2. Previous work in many labs has suggested that there is a fairly rigid requirement for the interactions of sigma domain 2.4 and 4.2 with their respective recognition elements at a distance of 10 and 35 base pairs from the start of transcription, respectively. We have previously demonstrated that polymerase lacking sigma70 domain 1.1 (sigma Del100) forms stable transcription complexes more slowly with some promoters, but much more rapidly from a promoter called Pminor. Pminor has poor matches to the expected ?10 and ?35 sequence elements. However, a consensus ?35 sequence (TTGAAA) is centered at ?28.5 relative to the Pminor transcriptional +1. To investigate whether this sequence is critical for Pminor expression, we constructed and assayed a series of Pminor deletions. Our results, in vitro with polymerases reconstituted using either full length sigma70 or sigma Del100, indicate that the TTGAAA sequence centered at ? 28.5 is required for Pminor activity, while the actual ?35 sequence is dispensable. In addition, altering the -28 region to perfectly match the consensus -35 sequence (TTGACA) greatly improved Pminor transcription by polymerase containing either full length sigma70 or sigma Del100. However, perfecting the -10 region (TATGGG to TATAAT) increased Pminor expression only when polymerase containing the full length sigma was used. Potassium permanganate footprinting assays were carried out to examine the Pminor transcription bubble. Although polymerase containing sigma Del 100 formed open complexes more readily than did polymerase with full length sigma, with either polymerase, the Pminor transcription bubble occurred farther downstream than is typical. In addition, slightly altered sensitivity patterns seen when the deleted sigma was present indicated that the promoter-polymerase architecture differs when domain 1.1 is missing. We conclude that polymerase recognizes Pminor by interacting with a -35 canonical sequence located at ?28.5 and that in the context of this "shorter" promoter, polymerase lacking domain 1.1 is better suited for open complex formation. Our results suggest that RNA polymerase has more flexibility in utilizing promoter sequences than has been previously recognized and that domain 1.1 of sigma 70 is involved in regulating this flexibility.