Methods of Walking with the RNA Polymerase

Background:The btuB riboswitch contains an unusual kissing loop structure that exhibits few interactions with bound adenosylcobalamin. Results: Structural probing and in vivo analysis show that kissing loop mutations strongly uncouple ligand binding from riboswitch regulation. Conclusion:The kissing loop is pivotal for coordinating riboswitch conformational changes. Significance: In contrast to most riboswitches, btuB relies on a kissing loop to achieve both metabolite sensing and gene regulation.

Section: Methodsmentioning

confidence: 99%

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

Lussier

Bastet

Chauvier

et al. 2015

“…Exonuclease Assays-His 6 -tagged Eco RNAP was immobilized on Co-nitrilotriacetic acid beads (Clontech) and walked along the linear DNA template labeled on the nontemplate strand to a desired position, as described previously (23). The halted ECs were probed with ExoIII (New England Biolabs) as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

Tagetitoxin Inhibits RNA Polymerase through Trapping of the Trigger Loop

Artsimovitch

Svetlov

Nemetski

et al. 2011

Self Cite

Background: Antibiotic tagetitoxin inhibits bacterial RNA polymerases (RNAPs) and RNAP III from eukaryotes. Results:We constructed a structural model of tagetitoxin bound to the transcription elongation complex. Conclusion: Tagetitoxin interacts directly with the ␤Ј subunit trigger loop, stabilizing it in an inactive conformation. Significance: Results have implications for designing new antibiotics and understanding principles of RNAP functioning and regulation.

“…Wild type Escherichia coli core polymerase was purified from cell biomass obtained from MRE600 strain as described (13). Yeast RNAP II was prepared as described (14).…”

Section: Methodsmentioning

confidence: 99%

Multisubunit RNA Polymerases Melt Only a Single DNA Base Pair Downstream of the Active Site

Kashkina

Anikin

Brueckner

et al. 2007

To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.Cellular RNA polymerases (RNAPs) 2 are multisubunit enzymes that share high structural and sequence homology from bacteria to eukaryotes. Based upon structural studies, it has been proposed that substrate NTPs might gain access to the active (insertion) site of these enzymes through two alternative routes. The first route is a relatively small opening (10 ϫ 10 Å) called the secondary channel (the "pore" in eukaryotic RNAPs) that leads directly to the active site and is a place where various transcription factors and inhibitors have been shown to bind. An alternative route may direct substrates through the main channel in the RNAP (formed by the two largest subunits), which binds the downstream DNA duplex. The differences in these substrate entry routes and the mechanisms of substrate loading are reflected in three major models. In the first model, the substrate NTP is believed to be loaded through the secondary channel into the insertion site either directly or following initial binding in the E-site, without preselection for the correct substrate base before its movement into the active site (1, 2). Another model suggests that the incoming substrate forms an incipient base pair with the templating base (n) in a preinsertion site before its movement to the insertion site. This scenario was demonstrated for T7 RNAP and a number of other nucleotide polymerases (3-5) and has also been proposed to operate in bacterial and yeast RNAPs (5, 6). Finally, the third model (the NTP lineup model) suggests that several (up to four) substrate NTPs may bind to the template DNA in the main channel before movement of the template base and its cognate substrate into the active site (7). The latter model would require that the downstream DNA be melted to provide an opportunity for base pairing with the substrate; however, the structural information available so far for the cellular RNAP ECs does not provide an unambiguous answer as to the extent of the downstream melting (2,6,8).In this work, we took advantage of nucleotide analogs whose fluorescence is sensitive to the local DNA environment (9 -11) to monitor melting of the downstream DNA. Our data demonstrate that cellular RNAPs melt only one DNA base pair downstream from the active site, a finding that is not consistent with the NTP lineup model, but favors a mechanism of substrate loading through the secondary channel one nucleotide at ...