Sharma M, Marshall P, Hinton D M
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
J Mol Biol. 1999 Jul 30;290(5):905-15. doi: 10.1006/jmbi.1999.2928.
During infection, the bacteriophage T4 transcriptional activator MotA, the co-activator AsiA, and host RNA polymerase are needed to transcribe from T4 middle promoters. Middle promoters contain a -10 region recognized by the sigma(70)subunit of RNA polymerase and a MotA box centered at -30 that is bound by MotA. We have investigated how the loss or modification of base determinants within the MotA box sequence 5'TTTGCTTTA3' (positions -34 to -26 of a middle promoter) affects MotA function. Gel retardation assays with mutant MotA boxes are consistent with the idea that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the C.G base-pair at position -30. In particular, the 5-methyl residue on the thymine residue at position -29, a major groove contact, contributes to MotA binding, while converting the T.A at -32 to a C. I base-pair, a change that affects the major but nor the minor groove, yields a MotA box that is similar to wild-type. However, methylation interference analyses indicate that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsXis inhibited by premethylation of guanine and adenine residues, suggesting that binding does not require minor groove contact with any specific T.A base-pair. Using gel retardation analyses, we calculate an apparent dissociation constant of 130 nM for MotA binding to the wild-type MotA box. Previous work has shown that the N-terminal region of MotA is needed for an interaction between MotA and sigma(70). We suggest that this MotA-sigma(70)interaction helps to stabilize the relatively weak interaction of MotA with the -30 region of middle promoter DNA.
在感染过程中，噬菌体T4转录激活因子MotA、共激活因子AsiA和宿主RNA聚合酶是从T4中间启动子进行转录所必需的。中间启动子包含一个由RNA聚合酶的σ（70）亚基识别的-10区域和一个位于-30处的MotA框，MotA与之结合。我们研究了MotA框序列5'TTTGCTTTA3'（中间启动子的-34至-26位）内碱基决定簇的缺失或修饰如何影响MotA功能。对突变MotA框进行的凝胶阻滞分析与以下观点一致：MotA利用中心GC上游的小沟接触和下游的大沟接触，并且在-30位的C.G碱基对处不需要任何特定的碱基特征。特别是，-29位胸腺嘧啶残基上的5-甲基残基（一种大沟接触）有助于MotA结合，而将-32位的T.A转换为C.I碱基对（一种影响大沟但不影响小沟的变化）会产生一个与野生型相似的MotA框。然而，甲基化干扰分析表明，鸟嘌呤和腺嘌呤残基的预甲基化既不抑制MotA与中间启动子PuvsXis的结合，也不抑制聚合酶/MotA/AsiA与中间启动子PuvsXis的结合，这表明结合不需要与任何特定的T.A碱基对进行小沟接触。通过凝胶阻滞分析，我们计算出MotA与野生型MotA框结合的表观解离常数为130 nM。先前的工作表明，MotA的N端区域是MotA与σ（70）相互作用所必需的。我们认为这种MotA-σ（70）相互作用有助于稳定MotA与中间启动子DNA的-30区域之间相对较弱的相互作用。
