Bacteriophage T4 Gene Expression

基因表达是一个高度受控的过程，对正常发育至关重要。在整个生物学中，这一过程可以通过转录起始位点的选择和转录起始的控制来调节。我们研究了一个简单的系统，大肠杆菌RNA聚合酶及其与T4噬菌体转录因子的相互作用，以了解影响转录起始的机制。大肠杆菌RNA聚合酶由包含RNA合成活性的核心和结合启动子序列设置转录起始的sigma因子组成。大肠杆菌的初级西格玛蛋白sigma70根据序列、结构和功能被划分为多个区域。区域1.1、2和4与依赖sigma70的启动子相互作用。在区域2中，alpha螺旋直接接触-10元素。在4区，sigma70的DNA表面很宽，4.1区有2个残基，4.2区有9个残基，沿着a ？35元素。与特定碱基的接触是通过区域4.2中的螺旋-旋转-螺旋。此外，区域4与核心的β -皮瓣结构之间需要接触来定位区域2.4和4.2，以便？10和？可同时接触35个元件。1.1区不与DNA相互作用，但影响DNA识别。在free sigma70中，1.1区阻止DNA结合。在全酶中，它调节稳定的启动子/聚合酶复合物的形成。

Gene expression is a highly controlled process that is crucial for normal development. Throughout biology, this process can be regulated through the selection of transcription start sites and the control of transcription initiation. We study a simple system, E. coli RNA polymerase and its interactions with transcription factors of bacteriophage T4, to discern mechanisms that affect transcription initiation. E. coli RNA polymerase is composed of a core, which contains RNA synthesizing activity, plus a sigma factor, which binds to promoter sequences, setting the transcription start. The primary sigma of E. coli, sigma70, is divided into regions based on sequence, structure, and function. Regions 1.1, 2, and 4 are involved in interactions with sigma70-dependent promoters. In region 2, an alpha helix directly contacts a -10 element. In region 4, the surface of sigma70 with DNA is extensive, involving 2 residues in region 4.1 and 9 residues in region 4.2 and extending for 9 bps along a ?35 element. Contact with specific bases is by a helix-turn-helix in region 4.2. In addition, contact between region 4 and the beta-flap structure in core is needed to position regions 2.4 and 4.2 so that the ?10 and ?35 elements can be contacted simultaneously. Region 1.1 does not interact with DNA, but it influences DNA recognition. In free sigma70, region 1.1 prevents DNA binding. In holoenzyme, it modulates formation of stable promoter/polymerase complexes. Regulation of the T4 life cycle is achieved largely by phage promoters, which sequentially express early, middle, and late genes. Because T4 does not encode its own RNA polymerase, it must direct the host transcriptional machinery to these phage promoters at the correct time. T4 accomplishes this takeover by encoding factors that alter the specificity of the host RNA polymerase as infection proceeds. T4 early promoters are active immediately after infection and contain the sigma70 ?10 and ?35 recognition elements. Middle promoters, which become active about 1 min after infection, contain the sigma70 ?10 element, but lack the sigma70 ?35 DNA element. Instead they have a MotA box sequence (5?atTGCTTtA3?) centered at ?30. Middle promoter activation requires both the T4 activator, MotA and a T4 co-activator, AsiA. AsiA is a 90 residue protein that binds tightly to sigma70 and works both as an inhibitor at sigma70-dependent promoters and as a co-activator at T4 middle promoters. MotA also interacts with sigma70 and binds to the MotA box. We have shown that sigma70 region 1.1 is not required for MotA/AsiA activation. However, the rate of AsiA inhibition and co-activation is significantly increased when region 1.1 is missing. We have also demonstrated that polymerase containing sigma70 with a region 1.1 deletion is significantly less stable than wild type polymerase. Previously, we have shown that AsiA-bound polymerase is generated by a two step process: AsiA first binds to region 4 of free sigma70 and then the AsiA/sigma70 complex binds to core. Our results suggest that in the absence of region 1.1, the dynamic equilibrium between polymerase and free sigma70 plus core shifts, yielding more free sigma70 at any given time. Thus, when region 1.1 is absent, the rate of AsiA inhibition of RNA polymerase lacking region 1.1 increases because of this increased availability of free sigma70. Structures of T. aquaticus and T. thermophilus RNA polymerase show that interactions between region 4 and the beta-flap position region 4.2 to interact with the ?35 element. Previous work has indicated that a substitution at residue F563 within region 4.1 affects sigma70 interactions with AsiA and with the beta-flap, suggesting that AsiA disruption of the sigma70/beta-flap interaction allows MotA to activate. We have tested the effects of mutations within sigma70 region 4 on sigma70 interaction with AsiA, MotA, and the beta-flap. Our results have shown that an alanine substitution patch at sigma70 residues 551, 552, 554, 555 severely impairs sigma70 transcriptional activity at sigma70-dependent promoters and interaction with AsiA and with the beta-flap. However, polymerase containing this mutant sigma is fully activated by MotA when AsiA is also present. In contrast, alanine substitutions at 557 and 560 render a sigma70 that behaves like wild type. In addition, sigma70 with a F563Y substitution is much less susceptible to AsiA inhibition, but is still fully activated by MotA/AsiA. Our results support the AsiA/sigma70 region 4 structure that shows AsiA contact with residues L551, R554, E555, and F563 but not with residues K557 or R560. Our results also provide evidence for a direct interaction between sigma70 region 4.1 and the beta-flap. Sigma70 region 4.2 residues R584, E585, and R588 have been implicated in directly contacting bps within the ?35 element. We have found that a sigma70 with alanine substitutions at these residues retains transcriptional activity but is much less concerned with the sequence of the ?35 element. This mutated sigma70 interacts poorly with AsiA. Polymerase containing this sigma70 is not inhibited by AsiA or activated by MotA/AsiA. Deletion of region 4.2 renders sigma70 immune to AsiA inhibition and MotA/AsiA activation. Our results suggest that the elimination of the sigma70 region 4.1/beta-flap interaction by the binding of AsiA is not sufficient to allow MotA activation. Consideration of our results in light of the AsiA/sigma70 region 4 structure argues that an interaction between AsiA and sigma70 region 4.2 is needed to present the far C-terminal region of sigma70 to MotA, facilitating the interaction between sigma70 and MotA.