Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Abstract

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ(70)-dependent (p1, pB, and p2) and two σ(S)-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models.

Fig. 1

Nucleotide sequence of the pcnB promoter region. The previously reported (or proposed) transcription start sites from particular promoters (pS1, p1, pB and p2) are marked, with first transcribed nucleotides indicated by large bold letters and arrows showing the direction of transcription, and −35 and −10 boxes are shown in frames. The Shine–Dalgarno (S.D.) sequences are underlined, and translation initiation codons (ATT and ATG, called +1(a) and +1(b), respectively) are indicated by bold letters

Fig. 2

Transcription from the pcnB (a–c), rsd (d) and fimB (e) promoter regions under various growth conditions of bacterial cultures. In experiments shown in a and b, wild-type (WT) E. coli or the pcnB (ΔpcnB::kan) mutant was grown in LB or MMGlu medium, to exponential (exp) or stationary (st) phase of growth at 37°C. Primer extension experiments were performed with primer Pr11 as described in “Materials and methods”, and the products of the reactions were separated during polyacrylamide gel electrophoresis, with the products of the sequencing reaction (performed using the same primer) run at the same gel (lanes G, A, T and C). The position of the products of primer extension reactions, corresponding to transcripts originating from the pB promoter, is shown in a. Positions previously reported as p1 and p2 transcription start sites are also marked by arrows. Apart from results of the experiment performed using the standard procedure (shown in a), results of a significantly longer exposition of the same gel during autoradiography are demonstrated (b) to show products corresponding to activities of other promoters. Analogous experiments, but with E. coli cells cultured at the exponential phase in LB medium (LB exp) and at the stationary phase (st) in various media (LB, MMGlu, MMGly, MMSuc, MMAce) are shown in c. In control experiments, levels of the rsd (d) and fimB (e) transcripts in E. coli wt cells (WT), and ppGpp⁰ (relA spoT double mutant), DksA⁰ (dksA::kan mutant) and ppGpp⁰ DksA⁰ (relA spoT dksA triple mutant) derivatives cultured at the stationary phase (d) or the exponential phase (e) in LB medium. Primer extension experiments were performed with Rsd.rev and FimB.rev primers, and the products of the reactions were separated during polyacrylamide gel electrophoresis, with the products of the sequencing reaction (performed using the same primer) run at the same gel (lanes G, A, T and C)

Fig. 3

Effects of ppGpp and DksA on the ompA gene expression (a) and the pB promoter activity (b, c). Wild-type (WT) E. coli or ppGpp⁰ (relA spoT double mutant), DksA⁰ (dksA::kan mutant) and ppGpp⁰ DksA⁰ (relA spoT dksA triple mutant) strains were cultured in the LB medium at 37°C to exponential (a and exp in b) or stationary (st in b) phase of growth. Primer extension experiments were performed as described in “Materials and methods”, and the products of the reactions were separated during polyacrylamide gel electrophoresis, with the products of the sequencing reaction (performed using the same primer) run at the same gel (lanes G, A, T and C). a and b show representative autoradiograms (positions of the products of primer extension reactions, corresponding to transcripts originating from ompA and pB promoters, are shown). c demonstrates quantification (by densitometric analysis) of the results exemplified in b (mean values from three independent experiments are shown in c with error bars indicating SD)

Fig. 4

Effects of DksA and ppGpp on in vitro transcription from the pB promoter. Representative results are shown on autoradiograms, and the summary of the results (mean values from three experiments with error bars indicating SD) is shown at the diagram (the presence of following factors in the reaction mixture is shown: diamonds DksA, squares ppGpp, triangles DksA and ppGpp). In control experiments, analogous reactions were performed with the λp _R promoter (the bottom panel), with either no additional factors (Ctrl), ppGpp (200 μM), DksA (400 nM) or ppGpp and DksA (200 μM and 400 nM, respectively)

Fig. 5

Effects of heat shock on levels of pB-derived transcripts in E. coli wild-type (WT) bacteria and the rpoD800 (rpoD) mutant. Bacteria were cultured to A ₅₇₅ of 0.4 (exponential phase) and then one half of the culture was transferred to 45°C (+) while the second half remained at 37°C (−). Following further cultivation for 15 min, RNA was isolated and primer extension experiments were performed as described in “Materials and methods”. The position of the products of primer extension reactions, corresponding to transcripts originating from the pB promoter, is shown

Fig. 6

Interaction of Eσ⁷⁰ with the promotor region of the pcnB gene as assessed by DNase I footprinting. A ³²P-labeled 0.3 kb DNA fragment was incubated with 0 nM (negative control, lanes 1 and 8), 10 nM (lane 2), 50 nM (lane 3), 100 nM (lane 4), 250 nM (lane 5), 500 nM (lane 6) or 1,000 nM (lane 7) Eσ⁷⁰. The footprinting experiments were performed as described in “Materials and methods”. The −10 and −35 sequences of p1, pB and p2 are indicated

Fig. 7

Effects of the presence of different (σ⁷⁰ or σ^S) sigma factors on in vitro transcription from pcnB promoters. Primer extension experiments were performed with primer pcnTR2 (as described in Fig. 2) using templates obtained in the in vitro transcription reactions, and either Eσ⁷⁰ or Eσ^S. Positions corresponding to transcripts derived from pS1, pB and p2 promoters are marked by arrows

Fig. 8

In vitro transcription from the region of the pcnB gene (a) and mapping of the pS2 transcription start sites (b). In experiments shown in a, E. coli RNA polymerase holoenzymes bearing different σ factors (marked above particular lanes) were used in the reactions performed as described in “Materials and methods”. Positions of transcripts derived from pB, pS1 and pS2 promoters are indicated. In the control experiment, activities of all holoenzymes were demonstrated by employing DNA templates containing promoters specific for various σ factors (templates with the rpoH gene promoter region and the dnaK gene promoter region, described by Janaszak et al. , were used); positions of bands corresponding for transcripts originating from particular promoters are indicated. In experiments shown in b, primer extension experiments with primer PS2.rev were performed using the products of in vitro transcription reactions as templates. In c, proposed localization of pS1 and pS2 promoters in the region upstream of the coding sequence of the pcnB gene is shown

Fig. 9

Activity of the pS1 promoter in vivo and its dependence on the rpoS gene function. Wild-type (WT) E. coli or ppGpp⁰ (relA spoT double mutant), rpoS and ppGpp⁰ rpoS (relA spoT rpoS triple mutant) strains were cultured in the LB medium at 37°C to exponential (exp) or stationary (st) phase of growth. Primer extension experiments were performed with primer PS1.rev as described in “Materials and methods”, and the products of the reactions were separated during polyacrylamide gel electrophoresis, with the products of the sequencing reaction (performed using the same primer) run at the same gel (lanes G, A, T and C)

Fig. 10

Activity of the pS2 promoter in vivo and its dependence on the rpoS gene function. Wild-type (WT) E. coli or ppGpp⁰ (relA spoT double mutant), rpoS and ppGpp⁰ rpoS (relA spoT rpoS triple mutant) strains were cultured in the LB medium at 37°C to exponential (exp) or stationary (st) phase of growth. Primer extension experiments were performed with primer PS2.rev as described in “Materials and methods”, and the products of the reactions were separated during polyacrylamide gel electrophoresis, with the products of the sequencing reaction (performed using the same primer) run at the same gel (lanes G, A, T and C)

Fig. 11

Effects of ppGpp and DksA on the pS1 (a) and pS2 (b) promoter activity. Wild-type (WT) E. coli or ppGpp⁰ (relA spoT double mutant), DksA⁰ (dksA::kan mutant) and ppGpp⁰ DksA⁰ (relA spoT dksA triple mutant) strains were cultured in the LB medium at 37°C to exponential (exp) or stationary (st) phase of growth. Primer extension experiments were performed as described in “Materials and methods”, with the use of PS1.rev primer (left panel) and PS2.rev primer (right panel), and the products of the reactions were separated during polyacrylamide gel electrophoresis, with the products of the sequencing reaction (performed using the same primers) run at the same gel (lanes G, A, T and C). Positions corresponding to pB, pS1 and pS2 transcription start sites are shown

Fig. 12

Effects of DksA and ppGpp on in vitro transcription from the pS1, pS2 (a–d) and λp _L (e–g) promoters. Representative results are shown on autoradiograms (a, b, e and f). The presence and concentrations of particular factors in the reaction mixtures are indicated and positions of bands corresponding to certain transcripts are shown. Summaries of the results (mean values from three experiments with error bars indicating SD) are shown at the diagrams (c, d and g). The presence of following factors in the reaction mixtures is shown: DksA (diamonds), ppGpp (squares), DksA and ppGpp (triangles)