Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination

Abstract

Transcriptional pause is essential for all types of termination. In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of mgtA and ribB riboswitches is controlled mainly by modulation of the stand-by rather than catch-up termination.

Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

“…It defines gene boundaries, silences production of useless and harmful RNA, and resolves R-loops 1 . Single-molecule fluorescence studies are consistent with the idea that ρ-dependent termination can proceed via two pathways 46 , 47 . In the classical RNA-dependent pathway, ρ binds C-rich, unstructured and unoccupied RNA regions via its PBSes; subsequent binding of a neighboring RNA region at the SBS and ring closure allow ρ to translocate towards elongating RNAP, eventually contact the transcriptional machinery around the RNA exit channel and terminate transcription 24 , 48 .…”

Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

“…It defines gene boundaries and silences production of useless and harmful RNA. In vitro , ρ-dependent termination can proceed via two pathways (45, 46). In the RNA-dependent pathway, ρ binds C-rich, unstructured and unoccupied RNA regions via its PBSes; subsequent binding of a neighboring RNA region at the SBS and ring closure allow ρ to translocate towards elongating RNAP, eventually contact the transcriptional machinery around the RNA exit channel and terminate transcription (47, 48).…”

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

“…Despite the difficulties in PIFE-FRET experiments where both dyes can be PIFE-sensitive, such as the combination of a Cy3 donor and a Cy5 acceptor, recent reports show the usefulness of this approach, at least in immobilised single-molecule assays, in sensing binding through both the donor dye and the acceptor dye, while reporting on conformational changes through FRET [144]. Recently, the quantitative interpretation of such donor-and-acceptor-PIFE-FRET experiments has been challenged through the use of a hidden Markov model approach suggested for the tandem analysis of both donor and acceptor PIFE changes and FRET changes [145].…”