Yang X, Price C W
Department of Food Science and Technology, University of California, Davis 95616, USA.
J Biol Chem. 1995 Oct 13;270(41):23930-3. doi: 10.1074/jbc.270.41.23930.
Rifampicin and streptolydigin are antibiotics which inhibit prokaryotic RNA polymerase at the initiation and elongation steps, respectively. In Escherichia coli, resistance to each antibiotic results from alterations in the beta subunit of the core enzyme. However, in Bacillus subtilis, reconstitution studies found rifampicin resistance (RifR) associated with the beta subunit and streptolydigin resistance (StlR) with beta'. To understand the basis of bacterial StlR, we isolated the B. subtilis rpoC gene, which encodes a 1,199-residue product that is 53% identical to E. coli beta'. Two spontaneous StlR mutants carried the same D796G substitution in rpoC, and this substitution alone was sufficient to confer StlR in vivo. D796 falls within Region F, which is conserved among the largest subunits of prokaryotic and eukaryotic RNA polymerases. Among eukaryotes, alterations in Region F promote resistance to alpha-amanitin, a toxin which inhibits transcription elongation; among prokaryotes, alterations in Region F cause aberrant termination. To determine whether alterations in the beta subunit of B. subtilis could also confer StlR, we made three StlR substitutions (A499V, G500R, and E502V) in the rif region of rpoB. Together these results suggest that beta and beta' interact to form an Stl binding site, and that this site is important for transcription elongation.
利福平与链霉溶菌素是分别在起始和延伸步骤抑制原核生物RNA聚合酶的抗生素。在大肠杆菌中，对每种抗生素的抗性源于核心酶β亚基的改变。然而，在枯草芽孢杆菌中，重组研究发现利福平抗性（RifR）与β亚基相关，而链霉溶菌素抗性（StlR）与β'亚基相关。为了理解细菌StlR的基础，我们分离出了枯草芽孢杆菌的rpoC基因，该基因编码一种1199个残基的产物，与大肠杆菌的β'亚基有53%的同源性。两个自发的StlR突变体在rpoC基因中携带相同的D796G替换，且仅这一替换就足以在体内赋予StlR抗性。D796位于F区域内，该区域在原核生物和真核生物RNA聚合酶的最大亚基中是保守的。在真核生物中，F区域的改变会导致对抑制转录延伸的毒素α-鹅膏蕈碱产生抗性；在原核生物中，F区域的改变会导致异常终止。为了确定枯草芽孢杆菌β亚基的改变是否也能赋予StlR抗性，我们在rpoB基因的利福平区域进行了三个StlR替换（A499V、G500R和E502V）。这些结果共同表明，β和β'相互作用形成一个Stl结合位点，且该位点对转录延伸很重要。
