Mapping the Spatial Neighborhood of the Regulatory 6S RNA Bound to Escherichia coli RNA Polymerase Holoenzyme

Abstract

Bacterial 6S RNA interacts specifically with RNA polymerase acting as transcriptional regulator. Until now, no detailed characterization of the spatial arrangement of the non-coding RNA within the three-dimensional structure of RNA polymerase has been performed. Here we present results obtained with the chemical nuclease FeBABE tethered to distinct positions of RNA polymerase σ70 subunit. 6S RNA complexes were formed with a collection of RNA polymerases, where the cleavage reagent had been fused to σ70 single-cysteine variants close to regions involved in promoter recognition. FeBABE-induced cleavage sites within the 6S RNA structure were identified, indicating close spatial neighborhood between σ70 single-cysteine side chains and defined positions of the 6S RNA structure. Our analysis demonstrates close proximity between the 6S RNA internal hairpin and σ70 domain 4.2, normally involved in recognition of −35 promoter DNA. Defined sections of the internal 6S RNA stem structure flanking the central bubble are positioned near conserved σ70 domains 3.1, 2.3 and 2.1, which are implicated in binding and melting DNA promoters between the −10 and −35 elements. Moreover, we show that U44 of 6S RNA is located near RNA polymerase active site (σ70 domain 3.2), fully consistent with its function as starting nucleotide in RNA-directed pRNA transcription. No neighboring contacts were detected between 6S RNA and σ70 region 1.2 or between σ70 and the 6S RNA closing stem structure (residues 1–41 and 144–184). Results were used to dock a structural model of 6S RNA to the known three-dimensional structure of Escherichia coli σ70 RNA polymerase holoenzyme.