Fig. 7
HSPA1B mutants affecting DNA secondary structure formation demonstrate differential pausing in vitro. a Plot shows free energy profile of HSPA1B gene around the in vitro pausing site. The difference in free energy (ΔG) between CGA mutant (less stable structure) and WT (CGC) is marked in red, while the ΔG difference between CGG (more stable structure) and WT is marked in green. Mutated sites are highlighted. In vitro transcription conditions (60 mM KCl, 7 mM MgCl2, and 30 °C) were used for Mfold analysis. b Structural probing of WT (CGC), CGG, and CGA with mung bean nuclease under in vitro transcription conditions. Each of the DNAs (30 nt) were treated with mung bean nuclease, which specifically cleaves single-stranded regions, and the products resolved on denaturing polyacrylamide gels. It showed that the CGG mutant displayed more protection at the region around the mutation (the affected region marked by a bracket and the mutation site marked by an asterisk), indicating the presence of double-stranded regions and thus the formation of hairpin structure. In contrast, the CGA mutant was more susceptible to the mung bean nuclease cleavage at the mutation site, suggesting the destabilization of the hairpin. c A representative polyacrylamide gel shows in vitro transcription of full-length (FL, 311-nt runoff transcripts) and paused (P) transcripts of wild-type HSPA1B, CGA, and CGG mutants, and no DNA template control in HeLa nuclear extracts. d The dependence of cumulative free energy difference and experimentally measured fraction of paused transcripts is shown. The cumulative free energy difference for each variant is determined from all possible secondary structures affected by the mutations (Additional file 1: Table S5). The line generated using robust linear regression (RLM function in Python) shows a correlation of strong RNA Pol II pausing associated with stable DNA secondary structures