Positive Supercoiling Ahead of RNA Polymerase Aids Exit from Protein-Mediated Loops

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.

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RNA polymerase: Preparation and structural analysis of helical polymers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010011091x ◽

1984 ◽

Vol 42 ◽

pp. 152-153

Author(s):

E. Loren Buhle ◽

Pamela Rew ◽

Ueli Aebi

Keyword(s):

Structural Analysis ◽

Rna Polymerase ◽

Molecular Level ◽

X Ray Diffraction ◽

Helical Polymers ◽

X Ray ◽

E Coli ◽

The Past ◽

Ordered Arrays ◽

Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

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Nutrient-regulated gene expression in eukaryotes

Biochemical Society Symposium ◽

10.1042/bss0730085 ◽

2006 ◽

Vol 73 ◽

pp. 85-96 ◽

Cited By ~ 8

Author(s):

Richard J. Reece ◽

Laila Beynon ◽

Stacey Holden ◽

Amanda D. Hughes ◽

Karine Rébora ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Control ◽

Direct Interaction ◽

Signalling Pathways ◽

A Cell ◽

One Step ◽

Higher Eukaryotes ◽

Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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