Journal: Nature structural & molecular biology
Article Title: RNA polymerase pausing and nascent RNA structure formation are linked through clamp domain movement
Figure Lengend Snippet: Modest inhibition of RNA:RNA duplex formation by RNAP ( a ) The ePEC nucleic-acid scaffold ( Table S1 ). DNA strands are shown in black, RNA in red, Pyrrolo-C (PC) in blue, and the 8-mer asRNA oligo in purple. The position of the pause (U19; −1 from 3′ end) is indicated by an arrow. ( b ) Models of E. coli RNAP EC and PEC. Left, closed-clamp, active EC model (based on PDB 2o5i and 3lu0 35 , 52 ) showing clamp (light pink), flap (light blue), bridge helix (BH; cyan), and location of major E.coli sequence insertions (SI1, SI2, SI3) 52 , 53 . The RNA:DNA hybrid (red and black), the exiting RNA (red), and asRNA (purple) are indicated. Right, model of open-clamp, duplex-stabilized PEC containing E. coli RNAP (based on PDB 4gzy 16 ) with arrows indicating clamp and flap movement upon duplex formation. The blow-up shows the location of the lid (pink), flap-tip, and PC (blue), which is quenched by duplex formation with asRNA in the PEC. ( c ) An averaged time trace of PC fluorescence ( n = 6) after mixing 1 µM asRNA with 250 nM ePECs. ( d ) asRNA concentration dependence of PC-quenching for ePECs. The slope of the linear regression line yields the average on-rate ( k on,ensemble ) of asRNA binding to ePECs (~80%) and free scaffold (~20%). ( e ) asRNA dissociation as monitored by PC fluorescence increase after addition of 100 µM competitor RNA to 500 nM preformed duplex-stabilized PEC (averaged trace; n=3). ( f ) The bimolecular rate constants of RNA duplex formation and asRNA dissociation in ePECs or free scaffold. Error bars, s.d. ( n = 3).
Article Snippet: Materials DNA and RNA oligonucleotides ( ) were obtained from IDT (Corvalville, IA); PC-containing oligonucleotides, from TriLink Biotechnologies (San Diego, CA); and 6-MI-containing oligonucleotides, from Fidelity Systems (Gaithersburg, MD).
Techniques: Inhibition, Sequencing, Fluorescence, Concentration Assay, Binding Assay