Mechanism of transcription initiation and promoter escape byE.coliRNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation byEscherichia coliRNA polymerase (RNAP; α2ββ’ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPRand T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.

Two transcription pause elements underlie a σ70-dependent pause cycle

The movement of RNA polymerase (RNAP) during transcription elongation is modulated by DNA-encoded elements that cause the elongation complex to pause. One of the best-characterized pause sequences is a binding site for the σ70 initiation factor that induces pausing at a site near lambdoid phage late-gene promoters. An essential component of this σ70-dependent pause is the elemental pause site (EPS), a sequence that itself induces transcription pausing throughout the Escherichia coli genome and underlies other complex regulatory pause elements, such as the ops and his operon pauses. Here, we identify and provide a detailed kinetic analysis of a transcription cycle analogous to abortive cycling that underlies the σ70-dependent pause. We show that, in σ70-dependent pausing, the elemental pause acts primarily to modulate the rate at which complexes attempt to disengage the σ70:DNA interaction. Our findings establish the σ70-dependent pause-encoding region as a multipartite element in which several pause-inducing components make distinct mechanistic contributions to the induction and maintenance of a regulatory transcription pause.