Journal: Nature Communications
Article Title: The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA
Figure Lengend Snippet: The Seb1-RRM domain has an unusual structure and RNA binding is essential. ( a ) Crystal structure of the Seb1-RRM 388–540 domain is shown as a cartoon representation, coloured in blue to red from the N- to the C-terminus. The position of the canonical RRM and the additional RRM-like domain is indicated below the structure. The two domains are interwoven and cross over between β2 and β3. ( b ) Electrostatic surface of the Seb1-RRM 388–540 shown in the same orientations as in a . Positively charged areas are coloured in blue and negatively charged areas in red. Arrows indicate a contiguous positively charged region tentatively assigned to interaction with the RNA phosphate backbone. ( c ) Plot showing a solution SAXS curve of the Seb1-RRM 388–540 (green). To compare the solution and crystallographic conformations of the Seb1-RRM 388–540 , a scattering profile was computed from the X-ray structure (black) and fitted to the solution scattering data. The quality of the fit as expressed as χ is indicated. ( d ) Flexibility analysis of the Seb1-RRM 388–540 (green) and a lysozyme standard (grey, BioisisID: LYSOZP) via dimensionless Kratky plot is shown. The intersection of the lines indicates the Guinier–Kratky point ( , 1.104), the peak position of an ideal globular and rigid protein. Rigid proteins show a characteristic parabolic shape with a peak at the indicated position (as is the case here), while unfolded proteins would plateau with increasing q -values. ( e ) Analysis of Seb1-SUMO-RRM 388–540 binding to FAM-tagged AUUAGUAAAA RNA by FA. Error bars indicate standard deviation of three technical replicates. ( f ) Spot test showing the effect of the indicated Seb1-RRM point mutations on cell growth.
Article Snippet: For RRM388–540 , binding was determined to 40 nM FAM-AUUAGUAAAA RNA (Eurofins) in 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2 , 1 mM DTT, 16.7% (v/v) glycerol, 0.1% IGEPAL, 0.1 mg ml−1 tRNA and 2.5% (v/v) RNasin (Promega).
Techniques: RNA Binding Assay, Binding Assay, Standard Deviation, Spot Test