Change in inorganic phosphate physical state can regulate transcription

ABSTRACT

Inorganic phosphate (P_i), a ubiquitous metabolite, is involved in all major biochemical pathways. We demonstrate that, in vitro, MgHPO₄ (the intracellular P_i form) at physiological concentrations can exist in a metastable supersaturated dissolved state or as a precipitate. We have shown that in solution, MgHPO₄ strongly stimulates exonuclease nascent transcript cleavage by RNA polymerase. We report here that MgHPO₄ precipitate selectively and efficiently inhibits transcription initiation in vitro. In view of the MgHPO₄ solubility and in vitro sensitivity of RNA synthesis to MgHPO₄ precipitate, at physiological concentrations, MgHPO₄ should cause a 50–98% inhibition of cellular RNA synthesis, thus exerting a strong regulatory action. The effects of P_i on transcription in vivo will, therefore, reflect the physical state of intracellular P_i.

Introduction

RNA polymerase (RNAP) transfers genetic information from DNA to messenger RNA in the course of transcription. This multi-step process [1,2] includes: (i) promoter recognition by RNAP holoenzyme (ii) initiation of RNA synthesis; (iii) elongation of RNA transcripts and (iv) termination, with release of full-size RNA transcript.

Initial binding of RNAP holoenzyme to the promoter is followed by melting of the DNA double helix. Melting enables RNA synthesis using one of the DNA strands as template. Short RNA oligonucleotides, synthesized in the transcription initiation complex (TIC) at the beginning of the transcription cycle, readily dissociate from the active centre, thus allowing RNA re-synthesis in cycles of abortive initiation. This process can repeat many times before the enzyme escapes to elongation, releasing σ subunit in the process of promoter clearance. After promoter clearance, the RNA/DNA/RNAP ensemble is dramatically stabilized as a ternary elongation complex (TEC). Advancement of TEC along the template, albeit processive, is frequently delayed by pausing. Some pauses are due to backtracking, that is, back-translocation of transcribing RNAP [3,4]. Backtracking unwinds the downstream edge of the RNA:DNA hybrid. The disengaged RNA 3′ segment occludes the active centre and prevents transcript elongation. This segment can be hydrolytically removed through RNAP endonuclease activity, which is greatly stimulated by bacterial factors GreA and GreB [5–7] and eukaryotic factor TFIIS [8], thus enabling transcript elongation from newly formed 3ʹ end. RNAP is also able to eliminate the 3′ terminal RNA residue in pretranslocated TEC by exonucleolytic cleavage [9].

Transcription is a highly regulated process (for review see [10–12]). To understand these regulatory mechanisms, we must elucidate how RNAP catalysis is altered by regulatory agents. These can be divided into i) protein factors, and ii) small molecules (e.g., cellular metabolites) that interfere with or enhance transcription. Representatives of the first class are more common, whereas there are few examples of cellular metabolites that directly target RNAP. Our in vitro data suggest that transcription can also be regulated by a novel mechanism through a change in the physical state of a cellular metabolite.

Inorganic phosphate, P_i, plays central roles in many cellular reactions [13–16]. Pi attachment to biomolecules mediates major cellular processes including differentiation, proliferation, cell death and survival, cell mobility as well as cell metabolism, intracellular signaling and energetics. Pi also serves as a buffering agent in mammalian cells and is a major component of bone and membranes. Depending on the organism, its functional state, and external conditions, among other considerations, intracellular P_i concentrations can vary over a wide range, ~5–100 mM [17,18]. Pi regulates RNA synthesis through association with protein factors that alter transcription from certain promoters by interacting with DNA [19,20].

At physiological pH, simple P_i salts are highly water-soluble. Thus, Na₂HPO₄ is soluble to ~0.5 M at ambient temperature. However, the intracellular Pi form, MgHPO₄, is poorly soluble at ~ 1.5 mM. Considering the high intracellular concentrations of P_i and Mg²⁺ and their ~1 mM complex formation constant, the bulk of intracellular P_i must exist as a precipitated complex with Mg²⁺. However, we demonstrated that the solubility equilibrium for MgHPO₄ requires prolonged incubation to establish, so that the kinetic solubility of MgHPO₄ formed in situ by mixing Na₂HPO₄ and MgCl₂ is much greater than its equilibrium solubility (see below). This permits formation of a metastable supersaturated solution, which could persist in the cell.

As we have shown previously [21], dissolved MgHPO₄ stimulates exonuclease nascent transcript cleavage by RNA polymerase nearly 2000-fold through engaging a Mg²⁺ ion in catalysis and activating an attacking water molecule at the RNAP active centre. Here we find that as a precipitate, MgHPO₄ strongly inhibits initiation of RNA synthesis in vitro. These distinct activities of the compound in different physical states suggest that MgHPO₄ could play a regulatory role in vivo. How it affects transcription in vivo would depend critically on its physical state in the cell.

Results

Determination of kinetic solubility of MgHPO₄

In experiments exposing an RNAP transcription initiation complex to MgHPO₄, we noticed an inhibition of RNA synthesis initiation that depended on how the compound was prepared. MgHPO₄ is poorly soluble in water. However, if prepared by mixing K₂HPO₄ with MgCl₂, MgHPO₄ starts to precipitate from a concentration of 20 mM (of each component) only after prolonged incubation at 20°C. This observation suggested that MgHPO₄ kinetic solubility was much higher than thermodynamic solubility. Therefore, to determine kinetic MgHPO₄ solubility, we generated MgHPO₄ by mixing the starting compounds at various concentrations. As seen from Figure 1, at concentrations 30 and 40 mM of each reactant, MgHPO₄ started to precipitate immediately, while at 20 mM, the solution remained transparent for many hours, suggesting that at this concentration MgHPO₄ formed a metastable supersaturated solution. Remarkably, the kinetic solubility of MgHPO₄ exceeds its equilibrium solubility at least 13-fold.

Figure 1.

Determination of the kinetic solubility for MgHPO₄. The turbidity of MgHPO₄ solutions generated by mixing the equivalent amounts of K₂HPO₄ and MgCl₂ at indicated concentrations.

Effect of MgHPO₄ on promoter-initiated full-size RNA synthesis

As seen in Figure 2(b) soluble MgHPO₄ had little effect on RNA synthesis initiated from the T7A2 promoter template (slots 1–5). However, if added as a precipitate, the compound significantly inhibited transcription at concentrations >2.5 mM (lanes 6–10; Figure 2(c)). The principal effect of insoluble MgHPO₄ was a general inhibition of RNA synthesis. In addition, RNA product distribution was affected. Increasing precipitated MgHPO₄ concentration significantly reduced the amount of 23 nt run-off transcript, but was less inhibitory on the synthesis of abortive 4–9 nt RNA products. The size-distribution within the latter group of products was similarly affected by precipitated MgHPO₄, resulting in the shift to smaller RNAs. Overall inhibition of RNA synthesis could be explained by disintegration of the RNAP open promoter complex, whereas the second effect – a relative increase in shorter transcripts at elevated [MgHPO₄] – could be due to interference with RNA elongation. Notably, we observed extended RNA products whose size, about 50 nt, exceeded that of the 23 nt template. Most likely, these products are generated upon template switching by RNAP [22]. The template switch RNA’s are decreased by both soluble and precipitated MgHPO₄.

Figure 2.

Effect of dissolved and precipitated MgHPO₄ on transcription from T7A2 promoter. (a). The scheme of the assay. (b). effect of various MgHPO₄ forms on promoter-initiated RNA synthesis. (c) Scans of the slots 6, 8–10 of B.

Inhibitory effect of precipitated MgHPO₄ is not promoter-specific

We then asked if RNA synthesis inhibition by MgHPO₄ precipitate was promoter-specific. To this end, we repeated the experiments with the T7A1 and ribosomal rrnB1 promoters. As seen in Figure 3, RNA synthesis was inhibited to the same extent on all templates, and is thus not promoter-specific.

Figure 3.

Effect of precipitated MgHPO₄ on transcription from various promoters.

Note the sharp drop off in RNA synthesis at 10 mM MgHPO₄; we discuss this point below.

Precipitated MgHPO₄ inhibits promoter-directed abortive dinucleotide synthesis

If RNA synthesis is initiated in the presence of an incomplete set of NTP substrates, stalled RNA products are released, and the initiation complex undergoes multiple abortive cycles. We tested the effect of precipitated MgHPO4 on abortive dinucleotide synthesis of pppGpC from GTP and [α-³²P]CTP at the T7A2 promoter. As seen in Figure 4, dinucleotide formation was also significantly inhibited. The response of this RNAP activity to precipitated MgHPO₄ was as robust as in the previous assay. This result is thus consistent with the proposal that precipitated MgHPO₄ disrupts an initiating RNAP complex.

Figure 4.

Effect of precipitated MgHPO₄ on abortive dinucleotide synthesis from the T7A2 promoter. (a). Reaction scheme. (b). Autoradiogram of the reaction mixture products after TLC separation. (c). Quantification of the results of B.

Precipitated MgHPO₄ does not significantly affect RNA elongation from a preformed TEC

To explore the effect of MgHPO₄ on transcription elongation, we exposed a reconstituted RNAP elongation complex, TEC11, to both forms of MgHPO₄ in the presence of 4 NTP substrates. Figure 5 shows that both dissolved and precipitated forms moderately inhibited RNA synthesis by retarding RNAP propagation (about 30 and 40%, respectively, at 20 mM MgHPO₄). This activity, however, was much smaller than that observed in the previous assays, supporting the idea that precipitated MgHPO₄ primarily inhibits an initiating complex rather than a TEC. TEC is much more stable than an open promoter complex, and therefore may be less susceptible to disruption.

Figure 5.

Effect of dissolved and precipitated MgHPO₄ on transcription elongation from assembled TEC. Reaction products were resolved by PAGE and visualized by phosphoimaging. Key reaction products are indicated.

Precipitated MgHPO₄ does not affect intrinsic nuclease transcript degradation by RNAP in TEC

As seen in Figure 5, a shorter RNA product was generated along with elongated transcripts. This dinucleotide product (pCpA) originates from TEC11 as a result of the intrinsic nuclease activity of the enzyme. Precipitated MgHPO₄ did not affect this reaction, again consistent with the notion that the agent selectively targets an RNAP initiation complex while having less effect on elongation and RNAP intrinsic transcript cleavage.

Precipitated MgHPO₄ inhibits RNA synthesis from a plasmid template

We next tested the effect of precipitated MgHPO₄ on RNA synthesis with a circular pTZ plasmid. This system permits multi-round RNA synthesis and monitors RNAP activity through a full transcription cycle. Figure 6 shows that as expected, MgHPO₄, also inhibited this reaction. Notably, the response of total RNA synthesis to precipitated MgHPO₄ was the same as that for abortive dinucleotide RNA synthesis, consistent with the primary effect of the agent on transcription initiation.

Figure 6.

Inhibition of RNA synthesis from a plasmid template by precipitated MgHPO₄. (a) Assay scheme. (b). Phosphoimage of the TCA-precipitated labeled RNA synthesized in the presence of MgHPO₄. (c). Quantification of the results of B.

Discussion

Solubility, a fundamental property of compounds, is defined as the maximal concentration of the compound in solution. Two distinct kinds of solubility exist: i) equilibrium solubility, a type of dynamic chemical equilibrium between a compound in the solid state and a solution of that compound, and ii) kinetic solubility, when the concentration of the compound in solution exceeds its equilibrium (thermodynamic) solubility. The later represents a supersaturated solution. Even though these solutions are metastable, some compounds do not precipitate over prolonged time periods. Thus, we find that the ubiquitous metabolite, inorganic phosphate, Pi, in the cellular form of MgHPO₄, can exist in vitro at physiologic concentrations in a supersaturated state, in which the MgHPO₄ concentration in solution significantly exceeds its equilibrium solubility, or as a precipitate. We discovered that in vitro MgHPO₄ in both these distinct physical states strongly modulates RNAP activity. In the metastable soluble form, MgHPO₄ stimulates RNAP intrinsic exoribonuclease activity on a TEC10 substrate [21], although less with other substrates, e.g., TEC11 (Figure 5). As a precipitate, MgHPO₄ inhibits initiation of RNA synthesis. Whereas stimulation of exonuclease can be rationalized by a structural model [21], how precipitated MgHPO₄ inhibits initiation remains unclear. Importantly, the surface of MgHPO₄ precipitates should contain densely arranged coordinated Mg²⁺ ions with free coordination valences. Inhibition of transcription initiation could, therefore, involve multiple interactions of RNAP open complexes with MgHPO₄ particles through these coordinations. These interactions could weaken attachment of the enzyme to promoter DNA, disintegrating the open complex. The insensitivity of the more stable TEC to precipitated MgHPO₄ is consistent with this notion.

Notably, inhibition of RNA synthesis as a function of the concentration of precipitated MgHPO₄ abruptly rose at compound concentrations > 5 mM (see Figures 2–4, and 6), reflecting a complex inhibitory mechanism. This could be explained by the presence of multiple inhibition targets in the RNAP initiating complex, residing on different sides of the enzyme surface. In this scenario, a granule of precipitated MgHPO₄ cannot interact simultaneously with more than one target. In such an arrangement, a small increase in granule concentrations will induce a disproportionally high inhibition, because the overall effect will be a composite of those caused by granules binding to each inhibitory site.

The properties of MgHPO₄ described above suggest that Pi could play a major role in cell biology. How MgHPO₄ effects transcription in vivo thus depends on its physical state in the cell, which, remarkably, has not yet been established. Life is a non-equilibrium process with respect to cellular energetics and concentrations of cellular metabolites [23]. Therefore, finding MgHPO₄ in a metastable supersaturated state in the cell would contribute to understanding the life processes by extending the concept of non-equilibrium nature of life to the state of a particular cellular metabolite. Finding MgHPO₄ as a precipitate would strongly suggest that the compound participates in transcription regulation by affecting RNA synthesis initiation.

Materials and methods

All chemicals were from Sigma-Aldrich. His₆-tagged RNAP was purified from E. coli strain RL721. Ultra-pure ribonucleoside-5ʹ-triphosphates and dATP were from Pharmacia. [α-³²P]CTP was from MP Biomed. Oligonucleotides were from Oligos. Radioactive products of polymerase reactions were resolved by electrophoresis in 15% polyacrylamide gel in the presence of 7 M urea, or by thin layer ion-exchange chromatography on PEI cellulose in 0.2 M KH₂PO₄ pH 4.0 developing system, and quantified by phosphoimagery using a Molecular Dynamics device (GE Healthcare).

Time course for precipitation of MgHPO₄ from supersaturated solutions

Equimolar amounts of K₂HPO₄ and MgCl₂ were mixed in 1 ml spectrophotometer cells to the desired concentration and kept at room temperature. Precipitation was monitored as increase in turbidity at 800 nm.

Preparation of supersaturated MgHPO₄

This was generated directly in the reaction mixtures containing transcription cocktails by mixing equimolar amounts of K₂HPO₄ and MgCl₂ to the desired concentration.

Effect of dissolved supersaturated MgHPO₄ on transcription from the T7A2 promoter

Reaction mixtures (10 µl) containing 120 nM RNAP, 40 nM T7A2 promoter, and 500 nM ϭ subunit were incubated 5 min at 37ºC and supplemented with K₂HPO₄ and MgCl₂ to the desired concentration, followed by addition of GTP, UTP, and ATP to final concentration of 0.2 mM and [³²P] CTP (30 Ci/mmol) to a final concentration 1 µM. After incubation for 15 min at 37ºC, the reaction was quenched by addition of an equal volume of 10 M urea, 10 mM EDTA, 0.05% Bromophenol Blue and 0.05% Xylene Cyanol dyes. The reaction products were resolved by denaturing 20% PAAG. The samples were then analyzed by electrophoresis (20% (wt/vol) PAAG, 7 M urea).

Preparation of precipitated MgHPO₄

0.5 ml of 0.4 M K₂HPO₄ was mixed with an equal volume of 0.4 M MgCl₂ and left for 1 h at room temperature.

Effect of precipitated MgHPO₄ on transcription from the T7A2 promoter

This was performed as in the case for dissolved supersaturated MgHPO₄, but the precipitated compound was added instead to pre-formed open RNAP-promoter complex prior to NTP addition.

Effect of precipitated MgHPO₄ on transcription from T7A1 and rnnB1 promoter

This was performed as described for T7A2 promoter by supplementing RNAP open promoter complexes with precipitated compound followed by NTP substrate addition.

Effect of precipitated MgHPO₄ on abortive dinucleotide synthesis from the T7A2 promoter

Reaction mixtures (9 µl) containing 80 nM RNAP, 40 nM T7A2 promoter, and 200 nM σ subunit in transcription buffer, TB (20 mM Tris⋅HCl, pH 8.0, 0.1 M NaCl, 10 mM MgCl₂) were incubated 5 min at 37ºC and supplemented with precipitated MgHPO₄ to the desired concentration The reaction was initiated by addition of 1 μl of a mixture of 2 mM GTP, 20 μM [α-³²P] CTP (specific activity 30 Ci/mmol). After 5 min of incubation at 37°C. the reaction was stopped by addition of 10 μl of 10 M urea with Bromphenol Blue and Xylene Cyanol dyes. Aliquots of the reaction mixture (1 μl) were applied to a PEI cellulose sheet (Sigma-Aldrich) and the reaction products were separated by thin-layer chromatography (TLC) using 0.5 M KH₂PO₄ as the developing solvent. The products were quantified by phosphoimagery.

RNAP elongation complex

TEC9A was assembled as previously described [21]. Briefly, RNA9 was mixed with equivalent amounts of DNA template strand to a final concentration of 1 µM in TB at 40°C and left to cool in a water bath to room temperature for ∼30 min. This mixture was added to an equivalent amount of His₆-tagged RNAP immobilized on NTA agarose, and kept at room temperature in a shaker for 10 min followed by addition of the equivalent amount of DNA nontemplate strand. The mixture was agitated for another 10 min at room temperature and supplemented with TB containing 1 M NaCl. After 10 min the pellet was separated and washed with regular TB. Labeled TEC10C, was obtained by elongation of TEC9A by an equivalent amount of [α-³²P] CTP (3000 Ci/mmol, 10 mCi/mL; MP Biomedicals) followed by washing with TB (4 × 1 mL). TEC11A was obtained from TEC10C by elongation with 10 µM ATP for 1 h at 0°C in TB containing 1 mM MgCl₂.

Effect of precipitated and dissolved supersaturated MgHPO₄ on elongation

A suspension of TEC11 on NTA beads in TB (8 µl) was supplemented with either precipitated MgHPO₄ or K₂HPO₄ and MgCl₂ to the desired concentration. This was followed by addition of 4 NTP substrates to a final concentration 40 μM and 10 min incubation at room temperature. The reaction was stopped by addition of 10 μl of 10 M urea with Bromphenol Blue and Xylene Cyanol dyes and the products analyzed by PAGE as described above.

Effect of precipitated MgHPO₄ on multi-round transcription from plasmid template

The reaction mixture (8 μl) containing 2 pmol RNAP, 0.05 pmol pTZ plasmid with a cloned T7A1 promoter and 2 pmol purified σ subunit TB was incubated for 5 min at 37°C and supplemented with 1 μl of 2 mM ATP, UTP, GTP and 0.4 mM [α-³²P]CTP (specific activity 30 Ci/mmol). After incubation for 30 min at 37°C the reaction was quenched by addition of 10 M urea and the aliquots (5 μl) were applied to Whatman 3 MM paper discs that were washed by 5% trichloroacetic acid (3 × 100 ml, 20 min each wash). The discs were rinsed with ethanol, dried and the remaining radioactive material quantified by phosphoimagery.

Funding Statement

This work was supported by Internal PHRI support grant to AM and by National Institutes of Health (grant number R01 GM037219) to MG.

Disclosure statement

No potential conflict of interest was reported by the authors.