The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli

Abstract

In Escherichia coli, stringently controlled genes are highly transcribed during rapid growth, but "turned off" under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like "stringent" RNAPs in the absence of the stringent response in vivo, constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.

Figure 1

In vitro transcription assays of rrnB P1 with wild-type (WT) and mutant RNAPs. The experiment was performed either without (lanes 1, 5, 9 and 13) or with an indicated inhibitor as described in Materials and Methods. The transcripts from rrnB P1 (≈170 nt), RNAI (≈110 nt), and tac are indicated.

Figure 2

Determining the stability of initiation complexes of rrnB P1 with wild-type (WT) and the mutant RNAPs. The experiment was performed with 50 mM KCl in the presence of competitive DNA pKK223–3 (≈80 nM) as described in Materials and Methods. Note different time scales for different experiments. The controls without the inhibitor are indicated as −. (A) Autoradiogram of the transcripts. (B) The transcription activities from rrnB P1 and RNAI were plotted as function of time (after inhibitor addition).

Figure 3

In vitro transcription assays of pyrBI with wild-type (WT) and mutant RNAPs. The experiment was performed with 50 mM KCl as described in Materials and Methods. Heparin concentration is indicated when it was present. The transcripts from pyrBI (≈135 nt) and P (≈325 nt) are indicated. (A) Transcription was performed with linear DNA, PvuII 758-bp fragment. (B) Transcription was performed with supercoiled DNA pBHM332.

Figure 4

Determining the stability of RNAP⋅pyrBI complexes under different conditions. Experiment was performed with 100 mM K-glutamate in the presence of heparin (10 μg/ml) as described in Materials and Methods. Note different time scales for different experiments. The controls without the inhibitor are indicated as −. (A) Wild-type (WT) RNAP on linear DNA (PvuII 758-bp fragment). (B) WT RNAP on supercoiled DNA pBHM332. (C) RpoB3449 on supercoiled DNA pBHM332.

Figure 5

In vitro transcription assays of P1 and P2 of the rpoD operon with wild-type (WT) RNAP and RpoB114. Transcription was performed as described in Materials and Methods. The transcripts form P1 (≈160 nt) and P2 (≈240 nt) of the rpoD operon and RNAI (≈110 nt) are indicated. (A) Transcription was performed on linear DNA (pDJ54 DNA that was digested with BamHI) with different concentrations of KCl (either with or without heparin). (B) Transcription was performed on supercoiled DNA pDJ54 with different concentrations of KCl in the absence of heparin. (C) Same as in B except that heparin was present.

Figure 6

Determining the stability of the complexes formed between RNAP and the P1 and P2 promoters of the rpoD operon. Experiments were performed with 50 mM KCl in the presence of heparin (100 μg/ml) as described in Materials and Methods. Note different time scales for different experiments. The controls without the inhibitor are indicated as −. Transcriptions were performed with wild-type (WT) RNAP on linear DNA (pDJ54 DNA that was digested with BamHI), and with WT RNAP and RpoB114 on supercoiled DNA pDJ54 as indicated.

Figure 7

A model of “transcription and the stability of initiation complexes are coupled at stringent promoters.” ITC, initially transcribing complex. EC, elongation complex. For details, see text.

Figure 8

The levels of IHF in wild-type (WT) and the rpoB114 mutant in the absence of the stringent response. IHF in exponentially grown cells were analyzed as described in Materials and Methods. The two subunits of IHF were indicated. Lane 1, size marker; lane 2, purified IHF; lane 3 and 4, wild type, but the amount of cells in lane 4 was 2-fold less than that in lane 3; lane 5 and 6, rpoB114, but the amount of cells in lane 6 was 2-fold less than that in lane 5. The cells in lane 3 and 5 were equal OD₆₀₀ units. Two different amounts of cells were used to ensure reproducibility.