Wu C W, Yarbrough L R, Wu F Y, Hillel Z
Biochemistry. 1976 May 18;15(10):2097-104. doi: 10.1021/bi00655a011.
sigma subunit of Escherichia coli RNA polymerase is known to stimulate specific RNA chain initiation. Rifampicin, an inhibitor of RNA chain initiation, binds to a single site on the beta subunit of RNA polymerase. We have used the fluorescence energy transfer technique to deduce proximity relationships of sigma subunit and rifampicin binding site on the enzyme. Isolated sigma subunit was covalently labeled with fluorescent donors in two ways: specific labeling of a single sulfhydryl residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate (1,5-I-AENS) and nonspecific labeling on the surface of the protein with dansyl chloride (Dns-Cl) adsorbed on Celite. The labeled sigma subunits were biologically active and formed a stoichiometric complex with core polymerase. The efficiency of energy transfer was obtained from the fluorescence intensity and the excited-state lifetime of the sigma-labeled holoenzyme in the presence and absence of rifampicin, which served as an energy acceptor. The transfer efficiency (2%) from AENS to rifampicin placed AENS somewhere between 42 and 85 A away from the rifampicin binding site. The rotational mobility of the donor was determined by nanosecond fluorescence depolarization spectroscopy, while the acceptor orientation was assumed to be fixed at some unknown angle. The efficiency measured for energy transfer from Dns to rifampicin was 10% in the presence of 0.2 M KCl. The distance from the surface of sigma subunit to the rifampicin binding site was calculated to be 27--38 A for a model having a randomly distributed and oriented array of donors on the surface of a spherical sigma subunit of 31-A radius. Our results indicate that rifampicin does not inhibit the initiation of transcription by RNA polymerase through a direct interaction with sigma subunit. In addition, energy transfer measurements under low salt conditions suggest that in RNA polymerase dimer the two rifampicin binding sites are symmetric with respect to each sigma subunit.
已知大肠杆菌RNA聚合酶的σ亚基能刺激特定RNA链的起始。利福平是一种RNA链起始抑制剂，它与RNA聚合酶的β亚基上的一个位点结合。我们利用荧光能量转移技术来推断σ亚基与酶上利福平结合位点的接近关系。分离出的σ亚基通过两种方式用荧光供体进行共价标记：用N-(碘乙酰氨基乙基)-5-萘胺-1-磺酸盐(1,5-I-AENS)对单个巯基残基进行特异性标记，以及用吸附在硅藻土上的丹磺酰氯(Dns-Cl)对蛋白质表面进行非特异性标记。标记后的σ亚基具有生物活性，并与核心聚合酶形成化学计量复合物。能量转移效率是通过在有和没有作为能量受体的利福平存在的情况下，σ标记的全酶的荧光强度和激发态寿命获得的。从AENS到利福平的转移效率(2%)表明AENS位于距离利福平结合位点42至85埃之间的某个位置。供体的旋转迁移率通过纳秒荧光去极化光谱法测定,而受体的方向被假定固定在某个未知角度。在0.2M KCl存在的情况下,从Dns到利福平的能量转移效率测得为10%。对于半径为31埃的球形σ亚基表面具有随机分布和取向的供体阵列的模型,计算出从σ亚基表面到利福平结合位点的距离为27-38埃。我们的结果表明,利福平不是通过与σ亚基的直接相互作用来抑制RNA聚合酶的转录起始。此外,低盐条件下的能量转移测量表明,在RNA聚合酶二聚体中,两个利福平结合位点相对于每个σ亚基是对称的。
