Journal: Nucleic Acids Research

Article Title: Fibrillarin/Nop1 perturbs RNA folding and assembly independently of liquid–liquid phase separation

doi: 10.1093/nar/gkag065

Figure Lengend Snippet: Nop1–rRNA interactions are displaced by assembly with ribosomal proteins. ( A ) Cy3-Nop1 (10 nM) binding to the 16S 5WJ rRNA was monitored by single-molecule colocalization. Ten nanomolar E. coli ribosomal proteins (RP) uS4 or uS7 were added to challenge Nop1. ( B ) Nop1 occupancy, defined as the fraction of time 5WJ rRNA is bound by Nop1. Orange symbols, average value over one trial (∼100 molecules); gray bars, average of independent trials. * P < .05 by one-way ANOVA. ( C ) Occupancy of Cy3-S4 on 5WJ rRNA with or without 10 nM unlabeled Nop1. The P -values were calculated by a one-tailed t -test. See for rastergrams.

Article Snippet: Proteins were overexpressed from plasmids and purified from E. coli BL21(DE3) (NEB; cat# C2527H) and Rosetta(DE3) (Novagen; cat# 71397-3) cells as described below.

Techniques: Binding Assay, One-tailed Test

Journal: Nucleic Acids Research

Article Title: Fibrillarin/Nop1 perturbs RNA folding and assembly independently of liquid–liquid phase separation

doi: 10.1093/nar/gkag065

Figure Lengend Snippet: Nonspecific Nop1 binding limits unwinding by CsdA. ( A ) Escherichia coli 5WJ rRNA tagged with ATTO 532 and ATTO 647N at its 3′ and 5′ ends has high FRET efficiency when folded . Unfolding by DEAD-box protein CsdA in 4 mM MgCl 2 and 1 mM ATP results in low FRET. Phase separation was initiated as in Fig. to investigate its effect on CsdA unwinding. ( B ) Change in average FRET efficiency for all 5WJ molecules. –Nop control: 200 nM CsdA was added at time 0. Inside and outside droplets, 200 nM CsdA was pre-mixed with Nop1 before phase separation. See for 2D histograms. ( C ) The change in rRNA FRET efficiency with or without 0.2 mg/ml tRNA. ( D – G ) 2 µM Nop1 was added without initiating phase separation, with or without 200 nM CsdA. ( H ) 200 nM Nop1 was pre-mixed with CsdA and added to the rRNA. ( I ) The rRNA was pre-incubated with CsdA, then 200 nM Nop1 plus CsdA was added at time 0. See for fitted parameters and confidence intervals.

Article Snippet: Proteins were overexpressed from plasmids and purified from E. coli BL21(DE3) (NEB; cat# C2527H) and Rosetta(DE3) (Novagen; cat# 71397-3) cells as described below.

Techniques: Binding Assay, Control, Incubation