Effects of DksA, GreA, and GreB on transcription initiation: insights into the mechanisms of factors that bind in the secondary channel of RNA polymerase

Abstract

Escherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfil some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentration of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When over-expressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA affected rRNA promoters only modestly in vitro and, even when over-expressed, GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the omega subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

Figure 1

GreB and DksA specifically inhibit transcription from rrnB P1 in vitro, enhance inhibition of transcription by ppGpp, and increase the iNTP concentration required for maximal transcription. (a) Effect of DksA, GreA or GreB (2μM) on transcription from rrnB P1. Single-round in vitro transcription reactions were carried out in duplicate in transcription buffer containing 30 mM NaCl with plasmid templates containing rrnB P1 (endpoints −61/+1; pRLG5944) (see Materials and Methods). RNA-I is a plasmid-encoded transcript. The amount of transcript from rrnB P1 in the presence of each factor relative to that with no factor is shown beneath the gel lanes. (b) Same as (a) except the plasmid contained the lacUV5 promoter (endpoints −46/+1; pRLG3422) (c) Effect of low concentrations of DksA, GreA or GreB on transcription from rrnB P1. Single-round in vitro transcription reactions were carried out in duplicate in transcription buffer containing 50 mM NaCl with plasmid template pRLG6555. Reactions contained 0.1 μM DksA, GreB, or GreA. (d) Effects of ppGpp and DksA, GreA, or GreB on transcription from rrnB P1. When indicated, reactions contained 100 μM ppGpp and 100 μM ppGpp with DksA (0.1 μM), GreB (0.1 μM) or GreA (6 μM). (e) Effect of DksA, GreA, or GreB on ATP concentration-dependence of transcription from rrnB P1. Multiple-round in vitro transcription reactions were carried out at increasing concentrations of the iNTP, ATP. The plasmid template was pRLG6555, and the buffer contained 170 mM KCl (see Materials and Methods). Transcription was normalized to the maximum (plateau) level for each condition. The DksA, GreA, and GreB concentrations were 3.5 μM, 3.5 μM, and 0.5 μM, respectively (see text).

Figure 2

DksA, GreB, and GreA reduce the RNAP-promoter complex half-life. (a) Half-life of the competitor-resistant rrnB P1 promoter complex in the presence of 0.1 μM DksA, 0.8 μM GreA, 0.1 μM GreB, or buffer only (no factor) was determined using a transcription based assay at 30ºC containing 30 mM NaCl and plasmid pRLG5944 (see Materials and Methods). Transcription at different times after addition of competitor (consensus promoter DNA) was normalized to the total amount of transcription at time zero. (b) Half-life of the competitor-resistant lacUV5 complex in the presence of DksA, GreA, or GreB, or buffer only (no factor) was determined using a filter binding assay at 25ºC in 100 mM NaCl (see Materials and Methods). The representative decay curves (0.75 μM of each factor) show the fraction of complexes remaining versus time after heparin addition. (c) Apparent affinities of each factor for RNAP. Half-lives of the competitor-resistant lacUV5 complex were determined at a range of DksA, GreA, and GreB concentrations or with no factor by the filter binding assay. The results are plotted as a function of the factor concentration. Best fit exponential decay curves were drawn using SigmaPlot. The inset shows an expanded view of the low concentration range. The apparent binding constants for each factor (i.e. the concentration of each factor needed to decrease the half-life of the complex by 50%) were ~0.1 μM DksA, ~0.1 μM GreB, and ~0.8 μM GreA. (d) – (f) Effects of DksA, GreB, and GreA on half-life in the presence of ppGpp. Half-lives of the competitor-resistant lacUV5 complex in the absence of factor, with 80 μM ppGpp alone, with 0.15 μM DksA (d), GreA (e), or GreB (f) alone, or with 80 μM ppGpp together with 0.15 μM DksA (d), GreA (e), or GreB (f). Half-lives were determined as in (b) (see also Materials and Methods) except the experiments were performed at 30ºC.

Figure 3

dksA, but not greA or greB, is required for regulation of rRNA synthesis in vivo. β-galactosidase activities (expressed in Miller Units) were determined in strains containing promoter-lacZ fusions. (a) – (d) rrnB P1 promoter (sequence endpoints −61 to +1; RLG5950). (e) and (f) lacUV5 promoter (−46 to +1; RLG5022). In (a), (b), (e), and (f), cells were grown in LB at 30ºC. In (c) and (d), cells were grown in M9 defined medium (see Materials and Methods) at 30ºC. (a), (c), (e) log phase (OD₆₀₀~0.4). (b), (d), (f) stationary phase. Numbers above each bar refer to the fold-change in promoter activity relative to the same promoter in the same growth condition in the wild-type strain. β-galactosidase activities from ≥ 2 cultures were averaged, and ranges or standard errors are shown.

Figure 4

DksA concentrations are higher than GreA and GreB concentrations at all points in the growth curve. Panels (a), (c), and (e) show representative Western blots from cell lysates of a wild-type strain (RLG5950) grown in MOPS defined medium (see Materials and Methods) in log phase (OD₆₀₀ = 0.4). Panels (b), (d), and (f) plot relative amounts of the factors versus OD₆₀₀ at different times in a growth curve. (Concentrations are expressed relative to DksA at OD₆₀₀ ~ 0.4, defined as 1.0). Standard curves made from blots of each purified protein were used to quantify the amounts of the factors in cell lysates. The DksA and GreB standards migrate slightly slower than the native proteins from cell lysates, because they contain hexahistidine tags. Concentrations (fmol factor/μg total protein) were determined from at least 3 separate experiments at each of six cell densities, as indicated. The absolute concentrations at OD₆₀₀ ~ 0.4 were: DksA, 137 ± 34 fmol/μg. GreA, 53 ± 17 fmol/μg. GreB, 13 ± 5 fmol/μg. Similar results were obtained for cells grown in LB medium (data not shown).

Figure 5

Overexpression of GreB, but not GreA, can partially compensate for the absence of DksA. In the indicated strain backgrounds, DksA, GreA, or GreB were expressed without IPTG induction from an lpp-lac promoter on plasmids derived from pINIIIAI (pRLG6333, pRLG8229, pRLG8242; see Materials and Methods; Table 1). Cells were grown in MOPS medium supplemented with 0.4% glycerol, 20 amino acids (see Materials and Methods), and 100 μg/ml ampicillin. (a) Protein concentrations, relative to the concentration of DksA in the wild-type (WT) strain, were determined by quantitative Western blots at OD₆₀₀ = 0.4 (log phase). Promoter activities were determined (b) in log (OD₆₀₀ ~0.4) and (c) in stationary phase (24 hr of growth). Strains were derivatives of RLG5950, carrying an rrnB P1-lacZ reporter on a λ prophage (see Materials and Methods). Averages and standard deviations were calculated from at least 3 experiments, except for the ΔdksA greA + pgreB strain, which was measured in duplicate. The number above each bar indicates the fold-increase relative to the rrnB P1 promoter activity of the wild-type strain in the same condition. (d) Doubling times in log phase in the MOPS medium used above were determined by monitoring OD₆₀₀ (wild-type, 56 min. ΔdksA, 88 min. ΔdksA + pdksA, 54 min. ΔdksA + pgreA, 93 min. ΔdksA + pgreB, 67 min. ΔdksA ΔgreA + pgreB, 66 min.)

Figure 6

ppGpp and DksA, but not the Gre factors, co-activate transcription at amino acid biosynthesis gene promoters. Multiple round in vitro transcription from Phis and PthrABC was carried out in duplicate in transcription buffer containing 165 mM NaCl and 2 μM DksA, 2 μM GreB, or 7 μM GreA with or without 100 μM ppGpp. The templates were (a) Phis (promoter endpoints −60 to +1; pRLG4413) or (b) PthrABC (promoter endpoints −72 to +16; pRLG5073). Transcription from duplicate lanes was averaged and is quantified as the ratio ± ppGpp. Each of the three factors increased transcription slightly in the absence of ppGpp, as described previously for DksA.

Figure 7

DksA, but not Gre factors, can compensate for the absence of the ω subunit of RNAP. (a) Single round in vitro transcription reactions were carried out in duplicate in transcription buffer with 30 mM NaCl using RNAP purified from a ΔrpoZ strain (see Materials and Methods). Reactions contained 0.5 μM DksA, GreA, GreB, or no factor and either 400 μM ppGpp or water. (b) and (c) Plasmid-encoded GreB cannot complement a strain lacking both dksA and rpoZ for regulation of rrnB P1. ΔdksA rpoZ (RLG8327) strains were transformed with an empty vector (pINIIIA1), pINIIIA1 encoding DksA (pdksA; pRLG6333), or pINIIIA1 encoding GreB (pgreB; pRLG8242). Cells were diluted into fresh LB with 100 μg/ml ampicillin at 30°C to an OD₆₀₀~0.025, and β-galactosidase activity was measured after 4 generations (OD₆₀₀~0.4) (b) or after 24 hr (c). The OD₆₀₀ after 24 hr was ~2.5 in the ΔdksA ΔrpoZ strain and ~5 in the other strains (see Results). Reported values are averages from at least 2 experiments.