MilliporeSigma
  • Cys-pair reporters detect a constrained trigger loop in a paused RNA polymerase.

Cys-pair reporters detect a constrained trigger loop in a paused RNA polymerase.

Molecular cell (2013-06-19)
Dhananjaya Nayak, Michael Voss, Tricia Windgassen, Rachel Anne Mooney, Robert Landick
ABSTRACT

Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Cystine, ≥98% (TLC), crystalline
SAFC
L-Cystine
Sigma-Aldrich
L-Cystine, ≥99.7% (TLC)
Supelco
L-Cystine, certified reference material, TraceCERT®, Manufactured by: Sigma-Aldrich Production GmbH, Switzerland
Sigma-Aldrich
L-Cystine, produced by Wacker Chemie AG, Burghausen, Germany, ≥98.5%
Cystine, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
L-Cystine, from non-animal source, meets EP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
Cystamine dihydrochloride, 96%
Sigma-Aldrich
Cystamine dihydrochloride, BioXtra
Sigma-Aldrich
Ribonucleic acid from torula yeast, core, Type II-C
Sigma-Aldrich
Ribonucleic acid from torula yeast, Type VI
Sigma-Aldrich
Ribonucleic acid from baker's yeast (S. cerevisiae)
Sigma-Aldrich
Ribonucleic acid diethylaminoethanol salt, Type IX
Sigma-Aldrich
Cystamine dihydrochloride, purum, ≥98.0% (AT)