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Mol Cell. 2013 Jun 27;50(6):882-93. doi: 10.1016/j.molcel.2013.05.015. Epub 2013 Jun 13.

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

Author information

1
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

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.

PMID:
23769674
PMCID:
PMC4037917
DOI:
10.1016/j.molcel.2013.05.015
[Indexed for MEDLINE]
Free PMC Article

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