Single-strand promoter traps for bacterial RNA polymerase

Abstract

Besides canonical double-strand DNA promoters, multisubunit RNAPs (RNA polymerases) recognize a number of specific single-strand DNA and RNA templates, resulting in synthesis of various types of RNA transcripts. The general recognition principles and the mechanisms of transcription initiation on these templates are not fully understood. To investigate further the molecular mechanisms underlying the transcription of single-strand templates by bacterial RNAP, we selected high-affinity single-strand DNA aptamers that are specifically bound by RNAP holoenzyme, and characterized a novel class of aptamer-based transcription templates. The aptamer templates have a hairpin structure that mimics the upstream part of the open promoter bubble with accordingly placed specific promoter elements. The affinity of the RNAP holoenzyme to such DNA structures probably underlies its promoter-melting activity. Depending on the template structure, the aptamer templates can direct synthesis of productive RNA transcripts or effectively trap RNAP in the process of abortive synthesis, involving DNA scrunching, and competitively inhibit promoter recognition. The aptamer templates provide a novel tool for structure-function studies of transcription initiation by bacterial RNAP and its inhibition.