Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator.
Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator.
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分析噬菌体 T4 AsiA 蛋白内涉及其与大肠杆菌 RNA 聚合酶 sigma70 亚基结合及其作为转录抑制剂和共激活剂的作用的区域。
DOI：
10.1016/s0022-2836(02)01307-4
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发表时间：
2003
影响因子：
5.6
通讯作者：
Hinton,DeborahM
中科院分区：
文献类型：
--
作者：
Pal,Debashis;Vuthoori,Madhavi;Pande,Suchira;Wheeler,David;Hinton,DeborahM
噬菌体T4 AsiA是一种由90个氨基酸组成的蛋白质，它与大肠杆菌RNA聚合酶的σ 70亚基结合，抑制宿主或T4早期转录，或与T4 MotA蛋白一起激活T4中期转录。为了研究AsiA内的哪些区域参与与σ 70形成复合物并提供转录功能，我们在整个AsiA中产生随机突变并在C末端区域内产生靶向突变。我们测试了突变蛋白在非抑制条件下补充T4 asiA am噬菌体生长、抑制大肠杆菌生长、在双杂交测定中与σ 70区域4相互作用、在天然蛋白凝胶中与σ 70结合以及在体外使用T4中间启动子抑制或激活转录的能力，所述T4中间启动子仅对RNA聚合酶有活性、被AsiA抑制并被MotA/AsiA激活。我们发现AsiA的N-末端一半内的氨基酸残基V14、L18和I40处的取代使得蛋白质与σ70的结合缺陷。这些残基位于AsiA二聚体的最近NMR结构中的单体-单体界面。相比之下，缺失C端44个氨基酸残基的AsiA在双杂交试验中与σ 70区域4良好地相互作用，并且缺失C端17个氨基酸残基（Δ74-90）的AsiA与σ 70结合并且在标准体外转录试验中完全胜任。然而，C-末端区域的存在延迟转录活性物种的形成时，AsiA/聚合酶复合物与启动子在MotA的情况下预孵育。我们的研究结果表明，氨基酸残基内的N-末端一半的AsiA参与形成或维持AsiA/σ 70复合物。AsiA的C-末端区域虽然不是抑制或共激活所绝对需要的，但通过减缓启动子和AsiA/聚合酶复合物之间的转录复合物的形成来帮助抑制。
Bacteriophage T4 AsiA, a protein of 90 amino acid residues, binds to the σ70subunit of Escherichia coli RNA polymerase and inhibits host or T4 early transcription or, together with the T4 MotA protein, activates T4 middle transcription. To investigate which regions within AsiA are involved in forming a complex with σ70and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region. We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under non-suppressing conditions, to inhibit E.coli growth, to interact with σ70region 4 in a two-hybrid assay, to bind to σ70in a native protein gel, and to inhibit or activate transcription in vitro using a T4 middle promoter that is active with RNA polymerase alone, is inhibited by AsiA, and is activated by MotA/AsiA. We find that substitutions within the N-terminal half of AsiA, at amino acid residues V14, L18, and I40, rendered the protein defective for binding to σ70. These residues reside at the monomer–monomer interface in recent NMR structures of the AsiA dimer. In contrast, AsiA missing the C-terminal 44 amino acid residues interacted well with σ70region 4 in the two-hybrid assay, and AsiA missing the C-terminal 17 amino acid residues (Δ74–90) bound to σ70and was fully competent in standard in vitro transcription assays. However, the presence of the C-terminal region delayed formation of transcriptionally competent species when the AsiA/polymerase complex was pre-incubated with the promoter in the absence of MotA. Our results suggest that amino acid residues within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/σ70complex. The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex.
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使用 T4 motA 蛋白和噬菌体修饰的 RNA 聚合酶从噬菌体 T4 中间启动子转录。
DOI：
10.1016/s0021-9258(18)55233-9
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
D. Hinton
通讯作者：
D. Hinton
噬菌体 T4 AsiA 蛋白与大肠杆菌的相互作用 σ
影响因子：
3.5
作者：
S. Pahari;D. Chatterji
通讯作者：
D. Chatterji
噬菌体 T4 AsiA 对大肠杆菌 RNA 聚合酶的抑制。
DOI：
10.1006/jmbi.1998.1742
发表时间：
1998
期刊：
Journal of molecular biology.
影响因子：
--
作者：
Severinova,E;Severinov,K;Darst,SA
通讯作者：
Darst,SA
影响因子：
3.6
作者：
Colland, F;Orsini, G;Kolb, A
通讯作者：
Kolb, A
老噬菌体，新见解：噬菌体 T4 发育中最近认识的两种转录调控机制。
影响因子：
2.1
作者：
E. Brody;G. Kassavetis;M. Ouhammouch;G. M. Sanders;R. Tinker;E. Geiduschek
通讯作者：
E. Geiduschek