Menendez M, Kolb A, Buc H
Institut Pasteur, Départment de Biologie Moléculaire, France.
EMBO J. 1987 Dec 20;6(13):4227-34. doi: 10.1002/j.1460-2075.1987.tb02771.x.
In Escherichia coli, the transcription of the malT gene is activated by the complex formed between cAMP and its receptor protein, CRP. Kinetics of formation of polyribonucleotide products from the corresponding promoter were studied in vitro by two sets of techniques, abortive initiation assays and run-off experiments. The first type of assay indicated that open complexes were formed at malT with an equivalent efficiency, and at comparable rates, whether CRP-cAMP was present or not. Secondary effects due to the activating complex were observed (increased stability of the open complex, elimination of a weaker binding site for the enzyme, improved Michaelis constants of RNA polymerase for the substrates of the assay, UTP in particular). But, primarily, CRP-cAMP did not exert a significant role in the rate of formation of the initiation complex. In contrast, run-off assays showed that the yield of the full-length transcripts was markedly enhanced by prior incubation of the DNA fragment with CRP-cAMP. Both in the presence and in the absence of activator, the rate-limiting step for this process was markedly slower than the formation of the initial open complex. Short oligonucleotides (n less than 9), probably arising from a recycling process, were found when the initiation complex was formed in the absence of CRP-cAMP. They were abolished by prior incubation with the activator. Unexpectedly, CRP-cAMP appears to favour the escape of RNA polymerase from the initiation complex at this promoter.
在大肠杆菌中，malT基因的转录由环磷酸腺苷（cAMP）与其受体蛋白CRP形成的复合物激活。通过两组技术（流产起始测定和延伸转录实验）在体外研究了从相应启动子形成多聚核苷酸产物的动力学。第一种测定类型表明，无论是否存在CRP-cAMP，在malT处均以相同效率和可比速率形成开放复合物。观察到了激活复合物产生的次要效应（开放复合物稳定性增加、消除了酶的较弱结合位点、改善了RNA聚合酶对测定底物尤其是UTP的米氏常数）。但是，主要的是，CRP-cAMP在起始复合物的形成速率中并未发挥重要作用。相比之下，延伸转录测定表明，通过将DNA片段与CRP-cAMP预先孵育，全长转录本的产量显著提高。无论是否存在激活剂，该过程的限速步骤均明显慢于初始开放复合物的形成。当在不存在CRP-cAMP的情况下形成起始复合物时，发现了可能来自循环过程的短寡核苷酸（n小于9）。预先与激活剂孵育可消除它们。出乎意料的是，CRP-cAMP似乎有利于RNA聚合酶在此启动子处从起始复合物中逃逸。
