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.

Graphical Abstract

Among the three compatible routes of ρ-dependent termination, riboswitch gating regulates the slowest route of the stand-by decomposing termination more predominantly than the two faster routes by the catch-up ρ.

Figure 1.

Fluorescent transcription complexes for single-molecule monitoring. (A) An exemplary fluorescent DNA template containing a single E. coli transcription unit with a ρ-dependent terminator. The template (gray) is labeled with a red-fluorescent Cy5 (magenta) at the downstream end for its real-time monitoring as well as with biotin (yellow) at the upstream end for its surface immobilization. (B) Its transcripts (blue) that are produced to have a greenish-yellow-fluorescent Cy3 (green) at the 5' end. When transcription elongation is resumed from the stalled transcripts, either termination occurs at the termination site (TS) to produce the terminated transcripts or readthrough takes place to yield the runoff transcripts. Both product transcripts have a rut site (orange) that ρ (not shown) can bind. (C) A fluorescent EC tied down on a microscope slide. A complex of RNAP, DNA and RNA with or without ρ is fixed on the polyethylene glycol (PEG)-coated surface through biotin-streptavidin-biotin conjugation. Individual complexes are spatially dispersed on the surface using a fraction of the biotin moieties each attached to only one in 41 PEG molecules.

Figure 2.

The three single-molecule assays with ρ-dependent terminators. (A) Left panel: the stand-by ρ assay's experimental scheme. PIFE occurrence of Cy3 (green) or Cy5 (magenta) is indicated by an enlarged circle. Right panel: the representative fluorescence time traces at Cy3 (top) and Cy5 (bottom) excitations exemplifying the stand-by decomposing termination route. NTPs were injected at 30 s (gray vertical line). (B) Left panel: the catch-up ρ assay's scheme. Right panel: the traces illustrating the catch-up decomposing termination route (top) and the catch-up recycling termination route (bottom). (C) Left panel: the ρ-free assay's scheme. Right panel: the traces demonstrating the readthrough runoff transcription. The delay between Cy3 PIFE diminishing and Cy5 PIFE appearance, denoted by t_run, corresponds to the elongation timespan without ρ.

Figure 3.

Measurements of pause and termination with ρ-dependent terminator templates. (A) Pause durations (t_p) of the five terminator templates. After the ρ-free elongation timespans (t_run) were separately measured with the mgtA (n = 387), rho (n = 1849), ribB (n = 1202), trp-t’ (n = 2409) and λ tR1 (n = 335) terminator templates and the NTP incorporation timespan (t_inc) was estimated for each template, their pause durations were calculated by t_p = t_run – t_inc. (B) Correlation between t_p and ρTE. The ρTEs of the stand-by decomposing (left), catch-up decomposing (middle) and catch-up recycling (right) termination routes were separately measured and are plotted on the y-axis against t_p on the x-axis. The Pearson's r, P-value and the fitting line slope (m) are shown. Error bar represents standard deviation of three independent datasets. The numbers of analyzed molecules are in Supplementary Table S2.

Figure 4.

Mutational analysis of the pause-termination correlations. (A) The eight mutations introduced to the mgtA terminator template around the major termination site (TS). The varied sequences are in lower cases. (B) The t_p values of the mgtA wild-type (WT) and mutant templates estimated as described in Figure 3A. (C) The correlation between t_p and ρTE analyzed as explained in Figure 3B. Error bar represents standard deviation of three independent datasets. The numbers of analyzed molecules are in Supplementary Table S2.

Figure 5.

Riboswitch ligand dependence of the three ρ-dependent termination route efficiencies. (A) Dependency of individual route's ρTE of the mgtA terminator on its riboswitch ligand Mg²⁺. The ρTEs of the stand-by decomposing (left graph), catch-up decomposing (middle graph) or catch-up recycling (right graph) route of mgtA on the y-axis are plotted against Mg²⁺ concentrations on the x-axis. Error bar represents standard deviation of three independent datasets. (B) Direct comparison of the individual route and total ρTEs between the riboswitch mostly closed at 2 mM Mg²⁺ or no ribocil-C and the one mostly open at 20 mM Mg²⁺ or 200 nM ribocil-C. (C) Plots of the ribB route ρTEs on the y-axis against ribocil-C concentrations on the x-axis similarly to Figure 5A. The numbers of analyzed molecules are in Supplementary Table S2.

Figure 6.

The pause-termination correlations in the presence of NusA/G, competitor RNAs or a crowder. The t_p and ρTE values of the five terminator templates were measured with addition of E. coli NusA/G factors (A), E. coli total RNA (B) or PEG-8000 (C). The recycling (solid) and decomposing (open) terminations were counted with each terminator in the stand-by (red) and catch-up (cyan) ρ assays. The ρTEs of the stand-by decomposing (left), catch-up decomposing (middle) or catch-up recycling (right) termination route are separately plotted on the y-axis against t_p on the x-axis. The Pearson's r, P-value and the fitting line slope (m) are shown. Error bar represents standard deviation of three independent datasets.The numbers of analyzed molecules are in Supplementary Table S2.

Figure 7.

Weighted effects of riboswitch gating on the three compatible ρ-termination routes. The ρ factor-dependent termination under riboswitch control or not proceeds via three different but compatible routes. The riboswitch gating by a ligand (green) most affects the stand-by decomposing termination (bottom row) among the three routes. Comparatively, the gating effects on the two catch-up routes (upper rows) are ancillary so simplified here to be absent. It is not known whether and how long ρ remains on the terminated transcript RNA. Moreover, the three routes operate on their distinct timescales, which is more evident here in Supplementary Figure S6 than our previous study (29). The stand-by ρ binds RNAP a priori earlier than the catch-up ρ. However, the stand-by ρ’s sole route for decomposing termination runs last as the slowest and is preceded by the catch-up ρ’s major route for decomposing termination in all the five terminators, although the timing error bars overlap in the rho terminator. Nevertheless, the catch-up ρ’s minor route for recycling termination comes clearly first as the fastest in all the terminators. The decomposing outcome is prevailing after ρ-dependent termination and would allow for 3D reinitiation by the reassociated RNAP (29). The recycling outcome renders the DNA-bound RNAP to diffuse on the same template molecule and lead to 1D reinitiation by the recycling RNAP just like after most intrinsic terminations, where the hairpin recycling route is much more frequent than the hairpin decomposing route (37,54).