RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases

Abstract

We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria-Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoB(H447R) and rpoB(D444G) prevent activation of the Prrn core promoter in rich medium, but only rpoB(H447R) also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoC(H113R) suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoC(P451L) prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication-transcription collisions. In a RecF(+) context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG.

Fig. 1

Two of the five rpo^sup RNA Pol mutations affect rrn expression in LB. β-galactosidase assays were performed on strains carrying a P_1rrnB-lacZ fusion and an rpo^sup mutation. The height of the histograms indicates β-galactosidase Miller Units, vertical bars indicate standard deviations. Wt stands for wild-type, Δ215–220 is the control rpoC^Δ215–220 mutation. P451L and H113R are rpoC mutations, D444G, H447R and N518D are rpoB mutations. Light grey: cells grown in MM; dark grey: cells grown in LB.

Fig. 3

A. rpo^sup RNA Pol mutations restore growth of rep uvrD cells. The height of the histograms indicates the number of colony-forming units (cfu) per ml, vertical bars indicate standard deviations. Rpo wild-type rep uvrD cells are not shown because they are lethal in all these conditions (plating on MM with casamino acids or on LB, at 37°C or 30°C). Mutants are in the AB1157 context, similar results were previously published for the rep uvrD rpoC^Δ215–220 mutant at 37°C in the MG1655 context (Boubakri et al., 2010). Δ215–220 and H113R are rpoC mutations, D444G, H447R and N518D are rpoB mutations. Light blue: MM 30°C (plating efficiencies on MM 37°C are not shown and were similar to those at 30°C); purple: LB 25°C; yellow: LB 30°C; orange: LB 37°C. Full boxes: colonies formed in 24 h (37°C LB), 48 h (30°C LB), or 3 days (30°C MM, 25°C LB). Hatched boxes: colonies appearing 24 h later than these normal times. B. Three rpo^sup RNA Pol mutations restore growth of rep uvrD dinG recF cells at all temperatures. Rpo wild-type and rpoC^P451L cells are not shown because they are lethal under these conditions (plating on MM or on LB, at 37°C or 30°C). Mutants are in the MG1655 context, similar results were obtained in the AB1157 context (not shown). Results for the rep uvrD dinG recF rpoC^Δ215–220 mutant at 37°C were previously published (Boubakri et al., 2010), and were reproduced here as a control. Plating efficiencies on MM 37°C are not shown and were similar to those at 30°C.

Fig. 2

A. Four rpo^sup RNA Pol mutations improve growth of UV-irradiated ruvABC mutants. Survival of UV-irradiated cells, results are the average of three to six independent determinations. Wild-type cells (JJC40) diamonds full line, ruvABC (JJC754) diamond dashed line, rpoC^Δ215–220ruvABC (JJC4886) crosses, rpoC^P451LruvABC (JJC4548) open square, rpoB^H447R ruvABC (JJC4832) closed circle, rpoB^D444G ruvABC (JJC4549) open circles, rpoB^N518D ruvABC (JJC4547) triangles. B. The rpoC^H113R mutation restores the viability of the ΔgreA::Cm^RΔgreB::Kan^R double mutant at 42°C. ΔgreA::Cm^RΔgreB::Kan^Rrpo^sup strains were streaked on LB Cm plates at 30°C (left) and at 42°C (right). Top sector, greA greB rpo^CH113R (JJC4923) the only thermoresistant greA greB double mutant. Turning in the clockwise direction from this mutant: greA greB rpoC^P451L (JJC5456), greA greB Rpo wt (JJC5455), greA greB rpoB^N518D (JJC4818), greA greB rpoB^D444G (JJC4820), greA greB rpoB^H447R (JJC4821).

Fig. 4

A. Three rpo^sup RNA Pol mutations restore growth of InvA dinG cells at all temperatures. The height of the histograms indicates the number of cfu per ml, vertical bars indicate standard deviations. Results for the InvA dinG rpoC^Δ215–220 mutant at 37°C were previously published (Boubakri et al., 2010), and were reproduced here as a control. Δ215–220, P451L and H113R are rpoC mutations, D444G, H447R and N518D are rpoB mutations (MG1655 context). Symbols are as in Fig. 3, dark blue are plating efficiencies on MM at 37°C. B. Three rpo^sup mutations restores growth of InvA dinG rep cells at 37°C on MM, only rpoB^D444G also restores viability on LB. Results for the InvA rep dinG rpoC^Δ215–220 mutant were previously published (Boubakri et al., 2010), and were reproduced here as a control.

Fig. 5

Schematic representation of the RpoB and RpoC subunits of RNA Pol showing the position of the rpo^sup mutations. In orange RpoB, in green RpoC. Blue and red lines represent the template and non-template DNA-strands, respectively, the pink line represents the neo-synthesized RNA (the putative backtracked RNA is shown in dashed pink line). Positions of the rpo^sup mutations are indicated (adapted with permission from Nudler, 2009).