title: Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue.
abstract: The secondary channel (SC) of multisubunit RNA polymerases (RNAPs) allows access to the active site and is a nexus for the regulation of transcription. Multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but the dynamics of these factors' interactions with RNAP and how they function without cross-interference are unclear. InEscherichia coli, GreB is an SC protein that promotes proofreading by transcript cleavage in elongation complexes backtracked by nucleotide misincorporation. Using multiwavelength single-molecule fluorescence microscopy, we observed the dynamics of GreB interactions with elongation complexes. GreB binds to actively elongating complexes at nearly diffusion-limited rates but remains bound for only 0.3-0.5 s, longer than the duration of the nucleotide addition cycle but far shorter than the time needed to synthesize a complete mRNA. Bound GreB inhibits transcript elongation only partially. To test whether GreB preferentially binds backtracked complexes, we reconstituted complexes stabilized in backtracked and nonbacktracked configurations. By verifying the functional state of each molecular complex studied, we could exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, GreB binds rapidly and randomly to elongation complexes, patrolling for those requiring nucleolytic rescue, and its short residence time minimizes RNAP inhibition. The results suggest a general mechanism by which SC factors may cooperate to regulate RNAP while minimizing mutual interference.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Tetone
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454.
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454.
authors.forename: Melisa L
authors.initials: ML
authors.lastname: Osborne
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454.
authors.forename: Harini
authors.initials: H
authors.lastname: Ravi
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.|Department of Bacteriology, University of Wisconsin, Madison, WI 53706.
authors.forename: Scotty
authors.initials: S
authors.lastname: Kyzer
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Stumper
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.|Department of Bacteriology, University of Wisconsin, Madison, WI 53706.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Mooney
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.|Department of Bacteriology, University of Wisconsin, Madison, WI 53706.
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454; gelles@brandeis.edu.
authors.forename: Jeff
authors.identifier: 0000-0001-7910-3421
authors.initials: J
authors.lastname: Gelles
mesh_terms: D001557:Benzenesulfonates; D001665:Binding Sites; D002232:Carbocyanines; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008958:Models, Molecular; D009010:Monte Carlo Method; D011485:Protein Binding; D000072760:Single Molecule Imaging; D013997:Time Factors; D061805:Transcription Elongation, Genetic; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000378:metabolism*
chemical_list: D001557:Benzenesulfonates; D002232:Carbocyanines; C495965:Cy3B N-hydroxysuccinimide ester; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C079667:GreB protein, E coli; D035602:Transcriptional Elongation Factors; C540081:rpoB protein, E coli; D012321:DNA-Directed RNA Polymerases
keywords: backtracking; secondary channel; total internal reflection fluorescence; transcription elongation