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Published on: October 1, 2017
RNA polymerase approaches its promoter without long-range sliding along DNA
Larry J Friedman1, Jeffrey P Mumm, Jeff Gelles
1Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
Summary
Escherichia coli σ(54)RNA polymerase does not use facilitated diffusion to find promoter DNA. Instead, direct binding from solution explains how this crucial enzyme efficiently locates its target sequences.
Area of Science:
- Molecular Biology
- Biophysics
- Genetics
Background:
- Sequence-specific DNA binding proteins must efficiently locate target sites within vast genomes.
- Facilitated diffusion (FD) is a proposed mechanism involving 1D sliding on non-target DNA.
- The search mechanism for RNA polymerases at promoter sites remains debated.
Purpose of the Study:
- To investigate whether Escherichia coli σ(54)RNA polymerase utilizes facilitated diffusion to find promoter sequences.
- To differentiate between facilitated diffusion and direct binding models for promoter recognition.
Main Methods:
- Colocalization single-molecule spectroscopy (CoSMoS) using multiwavelength fluorescence microscopy.
- Direct comparison of binding and dissociation rates to promoter and non-promoter DNA.
- Experiments with varying lengths of DNA flanking the promoter sequence.
Main Results:
- Initial binding to non-promoter DNA was significantly slower than to promoter-containing DNA.
- Altering the length of flanking DNA (7 bp to ~3,000 bp) did not affect the promoter-specific binding rate.
- These findings exclude facilitated diffusion over promoter-length distances as a significant search accelerator.
Conclusions:
- Facilitated diffusion is not the primary mechanism for σ(54)RNA polymerase promoter search.
- A direct binding mechanism, likely involving 3D diffusion in solution, is supported.
- Atypical DNA features at promoters may accelerate direct binding, explaining efficient target location despite potential DNA-bound competitors.
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