Effects of neutral salts on RNA chain elongation and pausing by Escherichia coli RNA polymerase.
We examined the effects of neutral salts and the non-ionic solute 2-methyl,-2,4-pentanediol (MPD) on transcript elongation by Escherichia coli RNA polymerase and on pausing induced by the multipartite his leader pause signal. All solutes tested slowed the overall rate of elongation, with anions showing the dominant effects in the order: (most inhibitory) HPO4(2-) > OAc- > SO4(2-) > ClO4- > I- approximately NO3- > Br approximately Cl- approximately MPD (least inhibitory). Although the protein structure-stabilizing anions HPO4(2-), OAc-, and SO4(2-) also increased the pause half-life at the his leader pause site, the remaining solutes accelerated escape from pause site in the order: (greatest acceleration) NO3- > ClO4- > I- > Br- > Cl- > MPD (least acceleration). Cl(-)-induced acceleration of escape from the pause site also occurred on mutant templates altered for the 3'-proximal region, RNA 3' end, or downstream DNA. The effect was eliminated, however, by base substitutions that destabilize the pause RNA hairpin or that extend it toward the 3' end. This "perfect hairpin" itself reduced the pause half-life by a factor of 3. We suggest that the pause RNA hairpin stabilizes a paused conformation of the transcription complex through an interaction with an easily disordered region of RNA polymerase. Extending the stem of the pause hairpin may disrupt the interaction by altering the position of the hairpin in the transcription complex. Anions may either compete for the interaction directly or disorder the site of hairpin interaction by chaotropic effects. We suggest that the negative effect of structure-stabilizing anions like OAc- and SO4(2-) may reflect passage of RNA polymerase through significantly different conformations during rapid elongation, some of which may expose hydrophobic surface.
Chan
C L
CL
Department of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63130, USA.
Landick
R
R
D000838:Anions; D001483:Base Sequence; D002712:Chlorides / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006018:Glycols / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011189:Potassium Chloride / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012492:Salts / Q000737:chemistry*; D014158:Transcription, Genetic
D000838:Anions; D002712:Chlorides; D006018:Glycols; D012492:Salts; D012313:RNA; D011189:Potassium Chloride; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C011480:hexylene glycol