RpaB, another response regulator operating circadian clock-dependent transcriptional regulation in Synechococcus elongatus PCC 7942

Abstract

The circadian clock of cyanobacteria is composed of KaiA, KaiB, and KaiC proteins, and the SasA-RpaA two-component system has been implicated in the regulation of one of the output pathways of the clock. In this study, we show that another response regulator that is essential for viability, the RpaA paralog, RpaB, plays a central role in the transcriptional oscillation of clock-regulated genes. In vivo and in vitro analyses revealed that RpaB and not RpaA could specifically bind to the kaiBC promoter, possibly repressing transcription during subjective night. This suggested that binding may be terminated by RpaA to activate gene transcription during subjective day. Moreover, we found that rpoD6 and sigF2, which encode group-2 and group-3 σ factors for RNA polymerase, respectively, were also targets of the RpaAB system, suggesting that a specific group of σ factors can propagate genome-wide transcriptional oscillation. Our findings thus reveal a novel mechanism for a circadian output pathway that is mediated by two paralogous response regulators.

FIGURE 1.

Analysis of RpaA binding with target promoters in vitro and in vivo. A, EMSA analysis with recombinant RpaA. ³²P-Labeled DNA fragments (50 fmol) of the promoter regions of rpoD6, radC, kaiBC, recB, rre36, smf, tetR, srrB, hik5, hoxU, rpoD5, or sigF2 were incubated in the absence (−) or presence (+) of 0.5 pmol of recombinant RpaA. B, ChIP-qPCR analysis. ChIP was performed with the anti-FLAG antibody and whole cell extracts of the Synechococcus RAF strain expressing RpaA-FLAG grown under continuous white light conditions (40 μmol of photons m⁻² s⁻¹). The amounts of immunoprecipitated DNA for the rpoD6, radC, kaiBC, recB, rre36, smf, tetR, srrB, hik5, hoxU, rpoD5, and sigF2 promoter regions were determined by qPCR analysis and are expressed as percent recovery relative to the total input DNA. Data are means ± S.D. of four amplifications from two independent experiments.

FIGURE 2.

Binding of RpaB to clock-dependent promoters in vitro. A–C, EMSA analysis with 0 (−), 1, 2, or 3 pmol of recombinant RpaA or RpaB as well as with ³²P-labeled DNA fragments (50 fmol) corresponding to the promoter regions of kaiBC (A), rpoD6 (B), or rpoD1 (C). The upper and lower bands correspond to DNA-protein complexes (C) and free probe (P), respectively. D and E, EMSA analysis for the promoter fragments of kaiBC (D) or rpoD6 (E) in the absence (−) or presence of recombinant RpaB (3 pmol) or 5- or 50-fold molar excesses of specific (kaiBC or rpoD6, respectively) or nonspecific (rpoD1) unlabeled DNA fragments as competitors. F, map of the kaiBC promoter region. Transcriptional initiation site is defined as +1. The putative HLR1 element is shown as an open box. Locations of competitors are shown as arrows 1, 2, and 3. G, EMSA analysis for the promoter fragments of kaiBC in the absence (−) or presence of recombinant RpaB (3 pmol) or 5- or 50-fold molar excesses of unlabeled, double-stranded oligonucleotides (#1–#3) as competitors.

FIGURE 3.

ChIP and qPCR analysis. ChIP was performed with antibodies to FLAG and with whole cell extracts of wild-type, RAF, or RBF Synechococcus strains (without FLAG, or with expression of RpaA-FLAG, or RpaB-FLAG, respectively) grown under continuous white light conditions (40 μmol of photons m⁻² s⁻¹). The amounts of immunoprecipitated DNA for the kaiBC, rpoD6, hliA, rpoD3, and rpoD1 promoter regions were determined by qPCR analysis and are expressed as percentage recovery relative to the total input DNA. Data are means ± S.D. of four amplifications from two independent experiments.

FIGURE 4.

Analysis of RpaB binding with target promoters in vitro and in vivo. A, EMSA analysis with recombinant RpaB. ³²P-Labeled DNA fragments (50 fmol) of the promoter regions of rpoD6, radC, kaiBC, recB, rre36, smf, tetR, srrB, hik5, hoxU, rpoD5, or sigF2 were incubated in the absence (−) or presence (+) of 3 pmol of recombinant RpaB. B, ChIP-qPCR analysis. ChIP was performed with the anti-FLAG antibody and whole cell extracts of the Synechococcus RBF strain expressing RpaB-FLAG grown under continuous white light conditions (40 μmol of photons m⁻² s⁻¹). The amounts of immunoprecipitated DNA for the rpoD6, radC, kaiBC, recB, rre36, smf, tetR, srrB, hik5, hoxU, rpoD5, and sigF2 promoter regions were determined by qPCR analysis and are expressed as percent recovery relative to the total input DNA. Data are means ± S.D. of four amplifications from two independent experiments.

FIGURE 5.

Inhibition of RpaB-promoter interaction by RpaA. EMSA analysis with various amounts of recombinant RpaA (0, 0.1, 0.5, 1, or 5 pmol) and with ³²P-labeled promoter fragments of kaiBC (A) or rpoD6 (B) in the absence or presence of recombinant RpaB (3 pmol).

FIGURE 6.

Oscillation of RpaB binding to the kaiBC and rpoD6 promoters. The time course of the binding of RpaA or RpaB to the kaiBC (A) or rpoD6 (B) promoter regions in RAF and RBF cells, respectively, maintained under LL conditions was examined by ChIP analysis of whole cell extracts. The amounts of DNA for each promoter region immunoprecipitated with antibodies to FLAG were determined by qPCR analysis and are expressed as percentage recovery relative to the total input DNA. Data are means ± S.D. of four amplifications from two independent experiments.