Figure 2.

(A) Proximity ratio histograms exhibit a sub-population with decreased transcription bubble size in RP_{ITC≤ 7}, which is reversible by addition of increasing amounts of GreA. Proximity ratio histograms of (−8)TA−(−5)NTD are shown. From top to bottom, histograms are shown for the conditions indicated in the labels of each panel. The solid black vertical line denotes the peak PR of a sub-population whereby the DNA between the dyes is annealed (either free promoter DNA or RNA-promoter closed complex, see vignettes). The dashed black vertical line denotes the peak proximity ratio the melted DNA in the open bubble sub-population in RP_{ITC = 2} (see vignette). The dashed red vertical line denotes the peak proximity ratio of a sub-population that is intermediate between the sub-populations of closed and opened bubble DNA. Sample preparation, measurement and analysis performed as explained by Ingargiola et al.,⁵⁹ only with triphosphate adenylyl(3′–5′) adenosine (pppApA, generously provided as a gift by Prof. Richard Ebright), instead of adenylyl(3′–5′) adenosine (ApA). Sub-population fitting analysis was performed as described in the appendix. (B) Two different paused initiation states as described by Duchi et al.¹ and by Lerner, Chung et al.,² and a model that combines both types of initiation pausing. RP_{ITC = 6, pre} and RP_{ITC = 6, post} denote RP_{ITC = 6} with the 3′-terminal base positioned at the pre- and post-translocated sites in RNAP, respectively. RP_{ITC = 7, pre} is defined in the same way as RP_{ITC = 6, pre}, but for a 7-mer nascent transcript. RP_{ITC = 7, 1 bt} denotes an initiation complex with a 7-mer nascent transcript backtracked by one base.