Use of RNA Polymerase Molecular Beacon Assay to Measure RNA Polymerase Interactions with Model Promoter Fragments

Abstract

RNA polymerase-promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase-promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase-promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase-promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.

Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA

“…T7A1cons is a consensus variant of the T7A1 promoter that is characterized by a low efficiency of promoter escape, in particular, in the case of RNAPs with mutations in σ region 3.2 ( 10 , 13 ). Since this promoter forms stable complexes with RNAP holoenzyme, we were able to measure their stability directly, by using the previously described molecular beacon assay ( 31 , 32 ). For this purpose, RNAP holoenzymes containing fluorescently-labeled σ 70 factor variants (either wild-type or 513–516A) were incubated with linear T7A1cons promoter templates, leading to a significant increase in the fluorescence intensity.…”

“…For T7A1cons, promoter complex stabilities were measured using a molecular beacon assay as described in ( 31 , 32 ). Promoter complexes were formed with tetramethylrhodamine-labeled derivatives of the wild-type or 513–516A σ 70 subunits (containing a unique cysteine residue introduced at σ position 211) in transcription buffer containing 40 mM Tris–HCl, pH 7.9, 10 mM MgCl 2 , 40 mM KCl and 0.01% Tween-20, and heparin was added to 100 μg/ml.…”

“…Promoter complexes were formed with tetramethylrhodamine-labeled derivatives of the wild-type or 513–516A σ 70 subunits (containing a unique cysteine residue introduced at σ position 211) in transcription buffer containing 40 mM Tris–HCl, pH 7.9, 10 mM MgCl 2 , 40 mM KCl and 0.01% Tween-20, and heparin was added to 100 μg/ml. After the addition of heparin, fluorescence intensity was measured every second, the relative fraction of the open complex was calculated by dividing the fluorescence intensity at each time point by the starting intensity before competitor addition, and the data were fitted to the one-exponential equation ( 31 , 32 ).…”

Reciprocating RNA Polymerase batters through roadblocks

“…Biotinylated RNAP holoenzyme. Biotinylated RNAP holoenzymes were reconstituted from a biotinylated RNAP core enzyme and σ 70 initiation factor, expressed as previously described 42,43 .…”