Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex

Abstract

A central enigma of transcriptional regulation is how the normally efficient transcription elongation complex stops at pause and termination signals. One possibility, raised by the discovery that RNA polymerase sometimes contracts its DNA footprint, is that discontinuous movements contribute to recognizing these signals. We report that E. coli RNA polymerase responds to sequences immediately downstream and upstream from the his leader pause site by changing neither its downstream DNA contact nor its upstream RNA contact for 8 bp preceding the pause. This compressed complex isomerizes to a paused conformation by an approximately 10 bp jump of its downstream DNA contact and simultaneous extrusion of an RNA hairpin that stabilizes the paused conformation. We suggest pausing and termination could be alternative outcomes of a similar isomerization that depend on the strength of contacts to 3'-proximal RNA remaining after the jump.

Pheromone cCF10 and plasmid pCF10‐encoded regulatory molecules act post‐transcriptionally to activate expression of downstream conjugation functions

“…The prgQ-R-S region is transcribed constitutively, but only the 5Ј inhibitor coding sequence or a closely coupled lacZ fusion is translated. An absence of translation complexes could lead to more frequent termination of transcription by RNA polymerase (Wang et al, 1995) and to rapid degradation of the transcribed region (Iost and Dreyfus, 1995) except for QS and the 3Ј portion of the prgS message. The prgTA transcripts lack the upstream prgS 3Ј UTR and boxA-like sequence (see below).…”

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

“…2B; supershifting of ␤ was similarly specific to Ϫ1 and pause; data not shown). Minus 1 and pause correspond exactly to the positions where weak and strong pausing, partial and complete hairpin formation, and stabilization against backtracking were observed previously (3,32). Thus, formation of the pause RNA hairpin in the paused complex appears to relocate the RNA away from ␤Ј into an unusual contact to ␤ that alters its gel mobility upon crosslinking.…”

“…The his leader pause hairpin is a well characterized nascent RNA structure that modulates RNA chain elongation (1)(2)(3)(4). It forms concomitantly with a paused conformation of the transcription complex midway through the his biosynthetic operon leader region, where it increases the lifetime of the paused complex at least 6-fold.…”

“…It is one of four components of the multipartite his pause signal that also depends on a 3Ј-proximal region of RNA or DNA, alignment of the 3Ј-terminal nucleotide and the incoming NTP, and duplex DNA that contacts polymerase downstream from the active site (1,2). Both the his pause signal and -independent terminators (which differ chiefly by shorter, U-rich 3Ј-proximal regions) destabilize DNA and RNA contacts in transcription complexes approaching the pause site, causing a propensity for backward sliding of the enzyme along the RNA and DNA chains (3,(17)(18)(19)(20). The potential for backtracking may slow translocation and thus dictate the position of hairpin formation (20); conversely, the hairpin, once formed, blocks backtracking in the rearranged complex.…”

“…In vitro transcription reactions using His 6 -tagged polymerase immobilized on Ni 2ϩ -agarose beads were performed as described previously (3). To incorporate 5-iodoUMP at positions U54-56 (Fig.…”