Systematic Studies of Effects of the Discriminator on Stabilization of the Open Complex and Abortive and Productive Initiation

E. coli RNA polymerase (RNAP) is a large molecular machine (subunits: αββ′ω and specificity factor σ 70 ) with many moving parts that binds to and opens duplex promoter DNA to initiate transcription. These events are set in motion by initial interactions between σ 70 and the −35 and −10 elements of the promoter, and between the flexibly tethered α‐CTDs and the UP element of the promoter. Binding free energy drives a series of conformational changes that culminate in opening of 13 base pairs between the −10 region and the start site, forming an initial unstable open complex, which is stabilized at some promoters by a network of interactions involving the discriminator and σ 70 region 1 in the cleft, and between the downstream duplex and downstream mobile elements (DME) of RNAP. Different promoter open complexes differ greatly in lifetime: the open complex at the rrnB P1 ribosomal promoter is very short‐lived (~1 s), as compared to 6 minutes for T7A1, and 17 hours for λP R promoter. To investigate effects of discriminator sequence and length on the structure, lifetime and function of the open complex, we are exchanging discriminator regions between promoters and determining KMnO 4 reactivities of open thymine bases, dissociation rate constants and patterns of abortive and productive initation using a common initial transcribed sequence. We extend previous findings by observing that the discriminator can be the primary sequence determinant of open complex lifetime; for example, inserting the T7A1 or rrnB P1 discriminator into λP R reduces its lifetime nearly to that of these promoters, while inserting the λP R discriminator into T7A1 increases its lifetime nearly to that of λP R . Footprinting reveals that template strand thymines upstream of the start site in the longer lived complexes have greater KMnO 4 reactivity. To interpret this, we propose that RNAP binds the phosphate backbone of this strand; tighter binding unstacks the bases more, as shown for SSB‐ssDNA by Lohman, and hence increases their KMnO 4 reactivity. To address the question of how the discriminator sequence and open complex lifetime affect abortive and productive initiation, we are conducting single round transcription assays on series of promoters differing only in the discriminator region. For the λPR series of discriminator variants, reproducible differences are observed in the amounts and pattern of both short and long products. Currently, research is focused on variants that exchange the λP R WT discriminator with those of rrnBP1 and T7A1 to determine how the discriminator region affects the distribution of short and long products as well as the absolute amount of products produced.