Transcription elongation. Heterogeneous tracking of RNA polymerase and its biological implications

Abstract

Regulation of transcription elongation via pausing of RNA polymerase has multiple physiological roles. The pausing mechanism depends on the sequence heterogeneity of the DNA being transcribed, as well as on certain interactions of polymerase with specific DNA sequences. In order to describe the mechanism of regulation, we introduce the concept of heterogeneity into the previously proposed alternative models of elongation, power stroke and Brownian ratchet. We also discuss molecular origins and physiological significances of the heterogeneity.

Keywords: Brownian ratchet; RNA polymerase; power stroke; sequence heterogeneity of DNA; transcription elongation factors; transcription pausing; translocation.

Figure 1. A model of sequence-specific pausing. (A) Pause-free elongation. RNA (orange), template DNA strand (gray), catalytic Mg²⁺ (magenta circle), and two RNAP domains (blue) involving 5′ RNA separation from the hybrid, i.e., Switch 3 (arrow head) and lid (triangle) domains are shown. The 3′ RNA-binding site (i) and the NTP binding site (i+1) are also indicated. (B) Elongation at the pausing site. The two sequence elements involved in transcription pausing are shown: (1) 3′ ACGC 5′ sequence in the transcribed DNA strand (grey) corresponding to the junction between the RNA-DNA hybrid and the downstream dsDNA in the elongation complex (indicated by shaded box); this sequence increases mobility/flexibility of the RNA/DNA backbones, which promotes fraying of the 3’ RNA end. (2) G residue in the RNA at the upstream end of the hybrid contributes to immobilization of the hybrid in the catalytic cleft of RNAP by interacting with the Switch 3 domain in the post-translocated state, or by interacting with the lid domain in the pre-translocated state.

Figure 2.Cis- and trans-acting factors affecting translocation. (A) Structure of RNA-DNA hybrid and dsDNA in TEC by T. thermophilus (Tth) RNAP. The structural targets for translocation regulators are indicated by arrows. (B) A schematic structure of TEC: RNAP (blue oval), upstream and downstream dsDNA (gray cylinders), RNA-DNA hybrid (brown cylinder), transcription bubble (black line) and the bridge helix (green) are shown; the active center in RNAP is represented by a circle with i and i+1 subsites. The inset displays the pre-translocated configuration of the active center with DNA lesion in i site (yellow triangle) and the 3′ RNA residue in a frayed configuration in i+1 site. The left side shows cis-acting translocation inhibitors: bending, or other structural alteration of the hybrid, the front-end DNA duplex and hairpin in the nascent RNA interacting with RNAP (shown by curved arrow). The right side displays the trans-acting inhibitors: a drug molecule bound to bridge helix reducing its mobility/bending (red dot), protein factors bound to dsDNA, nascent RNA or RNAP, and the second RNAP molecule in a head-to-tail configuration (all in magenta).

Figure 3. Translocation modulators target RNA-DNA hybrid and transcription bubble in bacterial TEC. Top panel: Nun protein of bacteriophage H022 interferes with translocation by stabilizing the -10 base pair of the hybrid and tethering RNAP to the hybrid. We proposed that the homologous N protein of bacteriophage λ stabilizes the -9 base pair of the hybrid and prevents the -10 base pair to favor translocation. E. coli NusG and its eukaryotic homolog Spt5 promote translocation by facilitating re-annealing of DNA immediately upstream from the 9-bp hybrid. B. subtilis NusG tethers RNAP to pre-translocated register by binding to the middle part of transcription bubble. σ⁷⁰ subunit interferes with translocation by binding to the “-10-like” sequence at the upstream end of transcription bubble. Bottom panel: cis-acting RNA hairpin and Switch 3 domain in β subunit promote translocation by preventing expansion of the hybrid to 10-bp length.