Large Effects Of Discriminator Exchanges On The Rna Polymerase-Promoter Open Complex Structure, Lifetime And Transcription Initiation Patterns

Escherichia coli RNA polymerase holoenzyme (RNAP; consisting of α2ββ’ω + σ70 subunits) is the molecular machine of transcription. This machinery is set in motion by initial recognition of −35 and −10 regions of linear promoter DNA by σ70 and of the UP element region by the two flexibly-tethered α-CTDs. A series of large conformational changes in both RNAP and DNA, driven by binding free energy, bend the downstream duplex into the active site cleft and open 13 base pairs including the −10 and discriminator regions and the transcription start site (+1). Subsequently at some promoters the initial unstable (lifetime 1 s) open complex is stabilized by a network of interactions involving the discriminator DNA, σ70 region 1.1, and downstream mobile elements (DME) of RNAP. These increase open complex lifetime to 6 minutes for T7A1 promoter and 17 hrs for the λPR promoter. An important but unanswered question is how the stability of the open complex affects its ability to initiate RNA synthesis upon binding of NTPs. Here we address this question by comparing lifetimes, structural features, and patterns of short and long RNA synthesized from λPR promoter with either the λPR or T7A1 discriminator, using nitrocellulose filter binding assays, permanganate footprinting and transcription assays. We also compare lifetimes and structural features of λPR , T7A1 and ribosomal rrnBP1 promoters with either λPR or T7A1 discriminator, as well as examine abortive and productive transcription products by acrylamide gel and novel mass spectrometric analysis to determine the extent to which the discriminator region is responsible for open complex lifetime, and to elucidate its effects on transcription initiation. This work is supported through funding by the NIH (GM103061).