Regulation of Gene Transcription

Cro对Lambda裂解启动子、PL和PR转录的激活：在表观遗传开关中的直接作用。如前所述，Lambda Cro蛋白通过表观遗传开关促进噬菌体的裂解生长。研究表明，在噬菌体感染或原噬菌体诱导后，Cro通过与OR3结合抑制PRM启动子来关闭CI合成。因此，在缺乏CI的情况下，裂解启动子将被抑制以帮助裂解生长。Cro以100 nM的C1/2与OR3结合。然而，在Cro关闭PRM后，通过SOS切割将CI水平降低到能够抑制PR和PL以促进噬菌体裂解生长的水平需要很长时间。现有的CI必须通过细胞分裂稀释出来，以便完全切换到噬菌体的裂解模式，即使在CI合成被Cro关闭之后。但是对PL和PR的镇压发生得更快。为了解释这一悖论，我们提出了即使在CI存在的情况下，Cro在帮助PL和PR转录中的直接作用。下面的结果证实了我们的想法。我们在体内和体外研究了CI和Cro存在下PR、PL和PRM的调控。虽然这些实验仍在进行中，但很明显，在120 nM浓度的CI抑制PR和PL 90%的条件下，即使极低浓度的Cro （75 nM）的存在也会导致PL和PR的显著抑制，这表明Cro的“遗传开关”更直接，而不是之前提出的间接模型。假设Cro和CI在诱导过程中的作用是相互独立的。我们发现在120 nM CI存在下，Cro增加了PL和PR。在体外，当低浓度的Cro同时存在时，与仅CI对照相比，裂解启动子变得活跃，表明Cro减轻了CI对PL和PR的抑制。在体内也是如此。当Cro蛋白从质粒转化到被抑制的前噬菌体时，与PR相关的lacZ报告基因抑制了β -半乳糖苷酶的合成，从macconkey -乳糖琼脂板上的红色可以判断。含有不含Cro质粒的等效菌株未观察到红色。这些范式转换的结果与Lambda原噬菌体诱导的遗传开关的经典理论相矛盾，表明Cro主要是通过失活CI抑制因子的作用来抑制裂解启动子PR和PL，而早在它关闭CI合成之前。在溶原性建立过程中，CI蛋白通过与OR2操作子结合激活PRM启动子。这是因为PRM结合的RNA聚合酶的sigma亚基与OR2结合的CI蛋白之间建立了接触。蛋白质与蛋白质的接触刺激了PRM转录起始的异构化步骤。令人惊讶的是，我们发现在这种接触中有缺陷的CI突变体（称为pc突变体）不仅不激活PRM，而且实际上抑制了PRM启动子的基础转录。到目前为止，我们的研究结果表明，抑制是通过pc突变体CI和RNA聚合酶之间的不同接触发生的，它们都与DNA结合。pc突变明显改变了RNA聚合酶的结构。提出的三元配合物的结构变化现在正在用低温电子显微镜进行测试。

Activation of transcription from Lambda lytic promoters, PL and PR by Cro: direct role in the epigenetic switch. As stated before, the Lambda Cro protein facilitates the phage's lytic growth by an epigenetic switch. It has been shown that Cro does so by turning off CI synthesis by repressing the PRM promoter by binding to OR3 after phage infection or prophage induction. Thus in the absence of CI the lytic promoters would be derepressed to help lytic growth. Cro binds to OR3 with a C1/2 of 100 nM. However, it takes a long time after the PRM turn-off by Cro to decrease the CI level by SOS cleavage to the level that would cause PR and PL derepression for facilitating phage lytic growth. The existing CI has to be diluted out by cell division for a full switch to lytic mode of the phage even after CI synthesis has been turned off by Cro. But repression of PL and PR happens sooner. To explain the paradox, we proposed a direct role of Cro in helping PL and PR transcription even in the presence of CI. Our idea has been corroborated by the following results. We studied the regulation of PR, PL and PRM in the presence of both CI and Cro in vivo and in vitro. Although these experiments are still in progress but it is clear that under the conditions in which CI at 120 nM concentration represses PR and PL 90%, the presence of even a very low concentration of Cro (75 nM) causes significant derepression of PL and PR establishing that the 'genetic switch' by Cro is more direct rather than the indirect model proposed earlier. It was assumed that the actions of Cro and CI during induction are independent of each other. We found Cro increases PL and PR in the presence of 120 nM CI. In vitro, the lytic promoters became active compared with CI-only control when a low concentration of Cro was simultaneously made available, suggesting that Cro alleviates the repression of PL and PR by CI. This is also true in vivo. When Cro protein was made in trans from a plasmid to a repressed prophage, a lacZ reporter gene linked to PR derepressed beta-galactosidase synthesis as judged by red color on MacConkey-lactose agar plates. No red color was observed from the equivalent strain harbors the plasmid without Cro. These paradigm-shifting results contradict the classical theory of the genetic switch in Lambda prophage induction, suggesting that Cro primarily causes prophage induction by inactivating the action of CI repressor to derepress the lytic promoters PR and PL long before it turns off CI synthesis. During the establishment of lysogeny, the CI protein activates the PRM promoter by binding to the OR2 operator. This happens because of an established contact between the sigma subunit of the PRM bound RNA polymerase and the OR2 bound CI protein. The protein-protein contact stimulates the isomerization step of the transcription initiation at PRM. Surprisingly we have found that a CI mutant defective in this contact (called pc mutant) not only does not activate PRM but actually represses the basal transcription of the PRM promoter. Our results so far indicate that the repression occurs by a different contact between the pc mutant CI and RNA polymerase both bound to DNA. Apparently the pc mutation has changed the structure of RNA polymerase. The proposed structural changes in the ternary complex is now being tested by cryo-electron microscopy.