Control Mechanisms In Temperate Bacteriophage

温带噬菌体的控制机制

• 批准号: 7594106
• 负责人: Robert A Weisberg
• 金额: $ 70.09万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594106
• 关键词: Bacteriophages; Base Pairing; Binding; Biological; Cells; Complex; DNA; DNA-Directed RNA Polymerase; Deoxyadenosines; Depth; Distant; Enzymes; Escherichia coli; Essential Genes; Gene Expression; Genes; Genetic Transcription; Goals; Hybrids; Laboratories; Length; Life; Localized; Modeling; Modification; Molecular; Movement; Nature; Nucleotides; Oligonucleotides; Pathway interactions; Polymerase; Positioning Attribute; Property; Proteins; RNA; RNA Binding; RNA Folding; Rate; Role; Site; Specificity; Structure; Surface; Thinking; Transcript; Uracil; Viral; Zinc; antitermination; base; deoxyadenosine; melting; mutant; novel; prevent; stem

HK022 uses a novel, RNA-based antitermination mechanism to express many essential genes. A nascent transcript of a viral sequence called put binds to the Elongation Complex (EC) that catalyzed its synthesis and remains associated with it as the transcript elongates. Association with put RNA modifies the EC so that it resists termination at downstream terminators. No other protein factor is required, other ECs in the same cell are unaffected, and there is no apparent terminator specificity. This elongation control pathway appears simpler than others, and its simplicity makes it an attractive target for deeper analysis. How does interaction with put RNA change the properties of the EC? Modification of the EC by put RNA increases the elongation rate and suppresses pausing at a uracil-rich sequence located immediately downstream of the put site. Since pausing is thought to precede termination, and some terminators contain uracil tracts, we decided to investigate the antipausing activity of put at this site. We have shown that the uracil-rich sequence promotes backtracking of the EC. Backtracking is a retrograde movement during which the most recently incorporated nucleotides (at the 3-prime end of the transcript) are melted from the template DNA strand and extruded from RNA polymerase, while compensating amounts of upstream RNA and DNA reenter the EC. We found that put RNA accelerates the EC through the uracil-rich pause by limiting the extent of backtracking. We considered three models of the relationship between the antitermination and antipausing activities of put. (1) Put RNA binds to the EC and then acts in the same way to suppress both pausing and termination. (2) Binding of put RNA to the EC is needed for suppression of termination but not of pausing. Instead, the bulky secondary structure of put RNA suppresses backtracking at the uracil-rich site by sterically inhibiting reentry of nascent RNA into the RNA exit channel of an EC. (3) Binding of put RNA to the EC is required for both antipausing and antitermination, but binding acts in different ways in the two pathways. To suppress pausing, binding anchors the nascent transcript to the EC so that it is unable to reenter the RNA exit channel (see below). To suppress termination, binding confers a persistent modification on the EC. The evidence presented next is consistent with model (3) but not with models (1) and (2). First, we showed that put efficiently suppressed terminators that are located as far as 10 kbp from the put site. By contrast, pausing at the uracil-rich sequence was no longer suppressed when we increased the put-pause distance by as few as 4 bp. Thus, the antipausing activity is local, but the antitermination activity is not. This difference argues strongly that the mechanism of antitermination differs from that of suppression of the uracil-rich pause, contrary to model (1). Second, put RNA folds into two stem-loops that are potentially capable of preventing backtracking by creating a local obstacle. However, studies with anti-put oligonucleotides and put mutants that should not perturb secondary structure argue strongly that formation of a folded structured is not by itself sufficient to prevent backtracking, contrary to model (2). In addition, an RNA polymerase mutant that is unable to bind put RNA is unable to suppress pausing at the uracil-rich sequence. We conclude that put suppression of backtracking at the uracil-rich pause site is the result of a local restrictive effect on retrograde RNA movement that is imposed by EC-bound put RNA. The restriction is relieved as the length of the transcript increases through further elongation. Put remains bound as the EC moves, and this presumably assures the persistence of antitermination. Backtracking of the EC is favored by thermodynamically weak RNA-DNA base pairs, such as that of the ribouridine-deoxyadenosine pair, and such weak hybrid is found at the termination points of intrinsic terminators. Our finding that put does not suppress pausing at distant uracil-rich sequences thus argues that put does not suppress termination by strengthening weak hybrid. The sharply localized nature of the antipausing activity of put allows us to estimate the distance between the end of the RNA exit channel of the EC and position to which put RNA binds. The uracil-rich pause site is located 21 nucleotides from the end of put RNA. Our evidence suggests that at this point, a put-modified EC can backtrack 2 nucleotides. (An unmodified EC can backtrack about 8 nucleotides.) It has been shown by others that about 14 nucleotides of newly synthesized RNA are retained within the EC, partly as RNA-DNA hybrid and partly as a single-stranded chain within the exit channel. These considerations suggest that there are 5 nucleotides (= 21 - 14 - 2) between the outside end of the RNA exit channel and the downstream end of bound put RNA at the point at which backtracking is completely prevented. If we assume that these 5 nucleotides are fully extended and lie along the surface of the EC, the end of bound put RNA is about 3 nm (= 5 nt x 0.6 nm/nt) from the end of the exit channel. The residues of RNA polymerase that are located at this distance include several that belong to a zinc-binding domain that is part of the beta-prime subunit. Mutational and RNA footprinting studies from our laboratory had previously identified this domain as a likely site of put RNA binding.

HK022使用一种新型的基于RNA的抗性机制来表达许多必需基因。 称为put的病毒序列的新生转录本与伸长复合物（EC）结合，该复合物催化其合成，并保持与转录物的伸长相关。 与PUT RNA的关联会修改EC，以便在下游终止者终止终止。 无需其他蛋白质因子，同一细胞中的其他EC不受影响，并且没有明显的终止特异性。 这种伸长控制途径似乎比其他人更简单，它的简单性使其成为更深入分析的吸引人目标。 与PUT RNA的相互作用如何改变EC的特性？ 通过放置RNA对EC进行修饰会增加伸长率，并抑制位于POT位点下游的富含尿嘧啶序列的暂停。 由于暂停是在终止之前的，并且一些终止者包含尿嘧啶区，因此我们决定研究该地点的PUT的反植物活性。我们已经表明，富含尿嘧啶的序列促进了EC的回溯。 回溯是一种逆行运动，在此过程中，最近掺入的核苷酸（在转录本的3次末端）从模板DNA链中熔化，并从RNA聚合酶中挤出，同时补偿了上游RNA和DNA reenter量的EC。 我们发现，将RNA通过限制回溯范围来通过富含尿素的停顿加速EC。 我们考虑了PUT的抗通过和抗抑制活动之间关系的三个模型。 （1）将RNA与EC结合，然后以相同的方式作用以抑制暂停和终止。 （2）将RNA与EC的结合是为了抑制终止而不是暂停。 取而代之的是，通过将新生的RNA重新进入EC的RNA出口通道，将RNA的庞大二级结构抑制了富含Uracil的位点的回溯。 （3）抗抑制性和抗通过抗性所需要的RNA与EC的结合是必需的，但是在这两种途径中结合以不同的方式起作用。 为了抑制暂停，将新生的转录本固定在EC上，以使其无法重新进入RNA出口通道（见下文）。 为了抑制终止，结合赋予对EC的持续修改。 接下来提供的证据与模型（3）一致，但与模型（1）和（2）不一致。 首先，我们表明，将有效抑制的终结者有效地抑制了位于PUT站点10 kbp的终结器。 相比之下，当我们将put puse距离增加到4 bp时，在富含尿嘧啶的序列处的暂停不再被抑制。 因此，抗脂肪活性是局部的，但抗授权活性不是。 这种差异强烈认为，抗授权的机理与抑制富含尿嘧啶的停顿的机制不同，与模型（1）相反。 其次，将RNA折叠到两个茎环中，它们可能能够通过创建局部障碍来防止回溯。 然而，对抗PUT寡核苷酸和放置突变体的研究不应扰动二级结构，强烈认为折叠结构化的形成本身不足以防止与模型相反（2）。 另外，无法结合pUT RNA的RNA聚合酶突变体无法抑制富含尿嘧啶的序列的暂停。 我们得出的结论是，将抑制在尿素富含尿素的暂停部位放置是对逆行RNA运动的局部限制性作用的结果，而RNA受到EC结合的RNA施加的逆行RNA运动。 随着转录本的长度通过进一步的伸长增加，限制会缓解。 随着EC的移动，PUT仍保持束缚，这大概可以确保抗授权的持久性。 热力学弱的RNA-DNA碱基对（例如ribouridine--脱氧腺苷对的）对EC的回溯进行回溯，并且在固有终止剂的终止点上发现了这种弱杂种。 因此，我们的发现并不能抑制在较远的尿素序列上停下来的暂停，因此认为PUT不会通过增强弱杂种来抑制终止。 PUT的抗抗病活性的局部局部性质使我们能够估算EC的RNA出口通道的末端与放置RNA结合的位置之间的距离。 富含尿嘧啶的暂停部位位于PUT RNA末端的21个核苷酸。 我们的证据表明，在这一点上，被改性的EC可以回溯2个核苷酸。 （未修饰的EC可以回溯约8个核苷酸。）其他人已经证明，大约14个新合成的RNA的核苷酸将保留在EC内，部分为RNA-DNA杂交，部分作为单链链，在出口通道内。 这些考虑因素表明，在RNA出口通道的外端和绑定的PUT RNA的下游端之间有5个核苷酸（= 21-14-2）。 如果我们假设这5个核苷酸已完全伸展并沿EC的表面位于EC的表面，则绑定的put RNA的末端从出口通道的末端开始约为3 nm（= 5 nt x 0.6 nm/nt）。 位于此距离的RNA聚合酶的残基包括属于Beta-Prime亚基一部分的锌结合域的几个。 我们实验室的突变和RNA足迹研究以前已经将该域确定为RNA结合的可能位置。