Regulation of Gene Transcription

Cro激活Lambda裂解启动子PL和PR的转录：表观遗传开关中的直接作用。如前所述，λ Cro蛋白通过表观遗传开关促进噬菌体的裂解生长。已经表明，Cro通过在噬菌体感染或原噬菌体诱导后结合OR 3来抑制PRM启动子，从而关闭Cl合成。因此，在不存在Cl的情况下，裂解启动子将被去阻遏以帮助裂解生长。Cro以100 nM的C1/2与OR 3结合。然而，在通过Cro关闭PRM之后，需要很长时间才能通过SOS切割将CI水平降低到将引起PR和PL去阻遏以促进噬菌体裂解生长的水平。现有的CI必须通过细胞分裂稀释，以便完全切换到噬菌体的裂解模式，即使在CI合成已经被Cro关闭之后。但是PL和PR的抑制发生得更快。为了解释这个悖论，我们提出了一个直接的作用，Cro在帮助PL和PR转录，即使在CI的存在。我们的想法已被以下结果所证实。我们研究了PR，PL和PRM在CI和Cro的存在下在体内和体外的调节。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，即使是非常低浓度的Cro（75 nM）的存在也会导致PL和PR的显著去抑制，这表明Cro的“遗传开关”更直接，而不是先前提出的间接模型。假设Cro和CI在诱导过程中的作用是相互独立的。我们发现Cro在120 nM CI存在下增加PL和PR。在体外，裂解启动子变得活跃相比，CI-只有控制时，同时提供低浓度的Cro，这表明Cro的抑制PL和PR的CI。在体内也是如此。当Cro蛋白从质粒到阻遏的原噬菌体反式制备时，与PR连接的lacZ报告基因解阻遏β-半乳糖苷酶合成，如通过MacConkey乳糖琼脂平板上的红色所判断的。从携带不含Cro的质粒的等同菌株中未观察到红色。这些范式转变的结果与λ原噬菌体诱导中遗传开关的经典理论相矛盾，表明Cro主要通过在其关闭CI合成之前很久使CI阻遏物的作用失活以解抑制裂解启动子PR和PL来引起原噬菌体诱导。在溶原性的建立期间，Cl蛋白通过与OR 2操纵基因结合来激活PRM启动子。这是由于PRM结合的RNA聚合酶的σ亚基与OR 2结合的Cl蛋白之间建立的接触而发生的。蛋白质-蛋白质接触刺激PRM处转录起始的异构化步骤。令人惊讶的是，我们已经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，抑制发生的pc突变体CI和RNA聚合酶之间的不同的接触都结合到DNA。显然pc突变改变了RNA聚合酶的结构。提出的三元复合物的结构变化，现在正在测试低温电子显微镜。

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.