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，这一过程可以通过选择转录起始点和控制转录起始来调节。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，以识别影响转录启动的机制。大肠杆菌RNA聚合酶由一个核心和一个西格玛因子组成，前者包含RNA合成活性，后者与启动子序列结合，设定转录起始。根据序列、结构和功能，大肠杆菌的初级西格玛(Sigma70)被分成不同的区域。区域1.1、2和4参与与依赖于sigma70的启动子的相互作用。在区域2中，α螺旋与-10元素直接接触。在第4区，Sigma70与DNA结合的表面很广，4.1区有2个残基，4.2区有9个残基，沿着一个？35单元延伸9bps。与特定碱基的接触在4.2区通过螺旋-旋转-螺旋进行。此外，为了定位区域2.4和4.2，需要在区域4和核心中的β-襟翼结构之间进行接触，以便可以同时接触？10和？35元件。区域1.1不与DNA相互作用，但它影响DNA识别。在游离型Sigma70中，1.1区阻止DNA结合。在全酶中，它调节稳定的启动子/聚合酶复合体的形成。 T4生命周期的调控在很大程度上是由噬菌体启动子实现的，噬菌体启动子依次表达早期、中期和晚期基因。因为T4不编码自己的RNA聚合酶，所以它必须在正确的时间将宿主转录机制引导到这些噬菌体启动子上。T4通过编码随着感染进行而改变宿主RNA聚合酶特异性的因子来完成这一接管。T4早期启动子在感染后立即激活，并含有Sigma70？10和？35识别元件。中间启动子在感染后1min左右开始激活，含有sigma70？10元件，但缺少sigma70？35DNA元件。相反，它们有一个MoTA盒序列(5？atTGCTTtA3？)中间价为？30。中间启动子的激活需要T4激活子MOTA和T4共激活子亚洲。ASIA是一个90个残基的蛋白质，与sigma70紧密结合，既是sigma70依赖启动子的抑制因子，也是T4中间启动子的协同激活因子。Mota还与sigma70相互作用，并与Mota盒结合。 我们已经证明，激活MOTA/Asia不需要sigma70区域1.1。然而，当区域1.1缺失时，亚洲抑制和共激活的比率显著增加。我们还证明了含有带有1.1区缺失的sigma70的聚合酶比野生型聚合酶显著不稳定。以前，我们已经证明了与亚洲结合的聚合酶是由一个两步过程产生的：亚洲首先与游离的sigma70的区域4结合，然后亚洲/sigma70复合体与核心结合。我们的结果表明，在没有1.1区的情况下，聚合酶和游离的sigma70+核心之间的动态平衡发生了移动，在任何给定的时间都会产生更多的游离的sigma70。因此，当区域1.1缺失时，由于游离态Sigma70可获得性的增加，缺少区域1.1的RNA聚合酶的亚洲抑制率增加。 水生假单胞菌和嗜热假单胞菌RNA聚合酶的结构表明，区域4和β-翻盖位置区域4.2之间的相互作用与？35元件相互作用。以前的工作表明，4.1区内残基F563的替换影响了sigma70与亚洲和与β-翻盖的相互作用，这表明亚洲对sigma70/β-翻盖相互作用的破坏允许MOTA激活。我们已经测试了Sigma70区域4的突变对Sigma70与ASIA、MOTA和β-Flat相互作用的影响。我们的结果表明，位于sigma70残基551、552、554、555的丙氨酸替代补丁严重损害了sigma70依赖启动子的转录活性，以及与亚洲和β-瓣的相互作用。然而，当亚洲也存在时，含有该突变西格玛的聚合酶完全被MOTA激活。相反，丙氨酸在557和560处的替换产生了行为类似野生型的sigma70。此外，具有F563Y替换的sigma70对ASIA抑制的敏感性要低得多，但仍可被MOTA/ASIA完全激活。我们的结果支持Asia/sigma70区域4结构，该结构显示亚洲与L551、R554、E555和F563残基接触，而不与K557或R560残基接触。我们的结果也为Sigma70区域4.1和β-瓣之间的直接相互作用提供了证据。Sigma70区4.2残基R584、E585和R588与？35元件内的BPS直接接触有关。我们已经发现，在这些残基上有丙氨酸取代的sigma70保留了转录活性，但与？35元件的序列关系不大。这个突变的sigma70与亚洲的相互作用很差。含有Sigma70的聚合酶不被ASIA抑制，也不被MOTA/ASIA激活。4.2区的缺失使Sigma70对ASIA抑制和MOTA/ASIA激活免疫。 我们的结果表明，通过ASIA的结合消除sigma70区域4.1/β-Flat的相互作用不足以激活MoTA。根据Asia/sigma70区域4的结构考虑我们的结果，认为亚洲和sigma70区域4.2之间的相互作用需要将sigma70的远C-末端区域呈现给MOTA，从而促进sigma70和MoTA之间的相互作用。

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.