Kim Hyeong C, Washburn Robert S, Gottesman Max E
Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA.
J Mol Biol. 2006 May 26;359(1):10-21. doi: 10.1016/j.jmb.2006.02.081. Epub 2006 Mar 29.
The 109 amino acid residue Nun protein expressed from prophage HK022 excludes superinfecting phage lambda by arresting transcription on the lambda chromosome near the lambdanut sites. In vitro, the Nun N terminus binds to nascent lambdanutRNA, whereas the C terminus interacts with RNA polymerase and DNA template. Escherichia coli host factors, NusA, NusB, NusE (S10), and NusG, stimulate Nun-arrest. NusA binds the Nun C terminus and enhances formation of the Nun-nutRNA complex. Because of these in vitro activities of NusA, and since a nusA mutation (nusAE136K) blocked Nun in vivo, we assumed that NusA was required for Nun activity. However, using a nusAts strain, we find that NusA is required for termination at nutR but not at nutL. Furthermore, nusAE136K is dominant to nusA(+) for Nun-arrest, both in vitro and in vivo. NusAE136K shows increased affinity for Nun and, unlike NusA(+), can readily be recovered in a ternary complex with Nun and nutRNA. We propose NusAE136K suppresses Nun-arrest when it is a component of the transcription elongation complex, perhaps, in part, by blocking interactions between the Nun C terminus and RNA polymerase and DNA. We also find that in contrast to Nun-arrest, antitermination by lambda N requires NusA.
来自原噬菌体HK022的109个氨基酸残基的Nun蛋白通过阻止λ噬菌体染色体上靠近λnut位点处的转录，来排除超感染的λ噬菌体。在体外，Nun的N端与新生的λnutRNA结合，而C端则与RNA聚合酶和DNA模板相互作用。大肠杆菌宿主因子NusA、NusB、NusE（S10）和NusG可刺激Nun介导的转录终止。NusA与Nun的C端结合，并增强Nun-nutRNA复合物的形成。鉴于NusA的这些体外活性，并且由于nusA突变（nusAE136K）在体内阻断了Nun的功能，我们推测NusA是Nun发挥活性所必需的。然而，利用一个nusA温度敏感型菌株，我们发现NusA是nutR位点转录终止所必需的，但不是nutL位点转录终止所必需的。此外，在体外和体内，nusAE136K对于Nun介导的转录终止而言相对于nusA（+）呈显性。NusAE136K对Nun的亲和力增加，并且与NusA（+）不同，它能很容易地在与Nun和nutRNA形成的三元复合物中被回收。我们提出，当NusAE136K作为转录延伸复合物的一个组分时，它会抑制Nun介导的转录终止，这可能部分是通过阻断Nun的C端与RNA聚合酶及DNA之间的相互作用来实现的。我们还发现，与Nun介导的转录终止相反，λ噬菌体N蛋白介导的抗终止作用需要NusA。
