Regulating Gene Expression Through DNA Mechanics: Tightly Looped DNA Represses Transcription.

DNA Mechanics;Gene Regulation;RNA Polymerase;Transcription;Cryo-Electron Microscopy;DNA Minicircles;Biomedical Engineering;Engineering (General);Mechanical Engineering;Biological Chemistry;Genetics;Molecular;Cellular and Developmental Biology;Physiology;Physics;Science (General);Engineering;Health Sciences;Science;Cellular & Molecular Biology

Lionberger, Troy AlbertWalter, Nils G. ;

University of Michigan

关键词: DNA Mechanics;    Gene Regulation;    RNA Polymerase;    Transcription;    Cryo-Electron Microscopy;    DNA Minicircles;    Biomedical Engineering;    Engineering (General);    Mechanical Engineering;    Biological Chemistry;    Genetics;    Molecular;    Cellular and Developmental Biology;    Physiology;    Physics;    Science (General);    Engineering;    Health Sciences;    Science;    Cellular & Molecular Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/75859/talion_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

It is now widely accepted that the mechanical state of DNA can play a major role in regulating the activity of RNA polymerase (RNAP). Not only have the global levels of supercoiling been shown to regulate transcription, but supercoiling has also been implicated in the transcriptional coupling of divergently oriented genes with closely spaced promoters. Additionally, many transcriptional repressors form tight loops of DNA by binding to multiple sites on a DNA template, challenging polymerases to transcribe a DNA template sustaining significant bending curvature. Many studies have provided evidence that the regulatory features of divergent promoter and loop-forming repressor systems share a dependence on the mechanical state of DNA, but these observations have been phenomenological in nature and fail to provide us with a mechanistic understanding of the relationship between RNAP activity as a function of the bending and twisting of DNA in these systems. Consequently, the direct role played by DNA mechanics in these systems remains unclear. I have hypothesized that the mechanical stress within highly bent DNA is itself sufficient to repress transcription. To test this hypothesis, I have developed an assay capable of quantifying the ability of bacteriophage T7 RNAP to transcribe small, circular DNA templates sustaining high levels of bending and torsional stresses. I have characterized both the pre-elongation and elongation kinetics using a highly untwisted 100 bp minicircle, an overtwisted 106 bp minicircle, and a mildly untwisted 108 bp minicircle template. In addition, I have used cryo-electron microscopy to directly observe the topological consequences of the torsional stress sustained within each DNA minicircle species at the single molecule level. Herein, I show that DNA minicircles on the order of 100bp can sustain significant torsional stress without relief by supercoiling, highly bent DNA is directly repressive to transcription, and torsional stress sustained within the DNA template modulates the elongation velocity and processivity of T7 RNAP. The data support a model in which DNA bending can directly control RNAP activity and call for more detailed studies to relate the mechanistic details emerging from this work to regulatory systems known to impart significant bends within the DNA template.