RNA Polymerase: Interaction of RNA and Rifampicin with the Subassembly α₂β

Abstract

We studied the inhibition of tryptic digestion of the subassembly x2/1 of Esclwichiu coli DNA-dependent RNA polymerase to investigate its interaction with RNA and rifampicin. Both agents decreased distinctly the cleavage of subunit lj in the subassembly as well as the degradation of the transiently formed polypeptides ( M , > 80000). Short RNAs with a chain length of approximately 35 nucleotides were most protective at a concentration of 1 m d m l while long. RNAs were less effective at the same concentration. DNA did not exert any observable prote&e effects. The association of RNA which separates z2/? bound to RNA from free rifampicin. The interpretation of data obtained with limited proteolysis is easier the fewer subunits there are involved. It has been observed that a stable intermediary subassembly is formed during the genesis of RNA polymerase holoenzyme from its subunits. This subassembly consists of only two kinds of subunits with the stoichiometry x 2 B [2,3] and, like the complete enzyme, binds rifampicin [4-61. RNA, the product of transcription, can inhibit the binding of rifampicin to this subassembly [7]. This observation suggested that 4 1 might also bind RNA. If so, it would be possible to study the interaction of RNA with the complex in the absence of subunit fl', which is the most basic subunit in the enzyme and binds polyanions such as heparin or DNA indiscriminately [8]. Furthermore the interference of RNA with the binding of rifampicin suggested the possibility that these two compounds might interfere competitively with each other in the binding to x2fl as a result of overlapping binding sites. We have, therefore, applied the method of limited proteolysis with trypsin to investigate the interaction of RNA and rifampicin with the subassembly x 2 / ? and the isolated subunit fi. In addition the association of RNA with clzfl and the competition by rifampicin were studied in binding experiments. MATERIALS A N D METHODS Nuclric AcidsUnlabelled poly(rU) and a mixture of 16-S + 23-S rRNA from Eschrrichiu coli were obtained from Boehringer Mannheim. Bacteriophage T7 DNA was a gift of Claudia Dubler. Yeast RNA was obtained from Sigma Chemie GmbH (Miinchen) (type XI RNA from bakers yeast, catalogue no. R-6750, prepared according to [9] E. coli RNA polymerase core enzyme, prepared according to [12], was separated into subunits [13]. The subassembly was formed immediately before use from the isolated subunits with a slight excess of cl [14]. The product was characterized by its mobility during gel electrophoresis in absence of detergent [15], which differs distinctly from that of the free subunits. The incorporation of fl into a2/? was essentially complete. Limited Tr~~,sinolysiLsThe reaction mix (total volume 0.11 ml) contained 0.1 nmol a2B, 10 mM Tris/HCI pH 7.9 (20"C), 165 mM KCI, 10 mM MgCI2, 0.1 mM EDTA, 0.25 mM dithiothreitol, 15"< ( V I V ) glycerol and additions as indicated. A stock solution of trypsin (Boehringer Mannheim) (I0 mg/ml in 0.05 M HCI) was diluted I : 5000 in reaction buffer immedia...

The β′ Subunit of Escherichia coli RNA Polymerase Is Not Required for Interaction with Initiating Nucleotide but Is Necessary for Interaction with Rifampicin

“…Early biochemical experiments demonstrated that, in agreement with our data, rifampicin derivatives cross-link to ␤ nonspecifically (20). On the other hand, it has been reported that ␣ 2 ␤ weakly binds Rif as judged by spectral, gel filtration-based, and proteolytic assays (21,22). However, in these later studies the authors did not determine whether their ␣ 2 ␤ preparation was free of traces of the RNAP core and whether the observed interaction was specific because no controls using ␣ 2 ␤ containing the ␤ subunit carrying a Rif resistance mutation were used.…”