In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme

doi:10.1093/nar/gkq377

Nucleic Acids Res. 2010 July; 38(13): e141.

Published online 2010 May 10. doi: 10.1093/nar/gkq377

PMCID: PMC2910072

In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme

Haruichi Asahara and Shaorong Chong^*

New England Biolabs Inc., 240 County Road, Ipswich, MA 01938, USA

*To whom correspondence should be addressed. Tel: Phone: +1 978 380 7324; Fax: +1 978 921 1350; Email: chong/at/neb.com

Received March 3, 2010; Revised April 23, 2010; Accepted April 26, 2010.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

This article has been cited by other articles in PMC.

Abstract

In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes.

INTRODUCTION

Advances in recombinant DNA technologies have allowed in vitro biochemical studies of recombinant proteins with important cellular functions. Often the most daunting and time-consuming studies involve purifying and assembling multiple, sometimes a large number of, recombinant proteins for in vitro reconstitution of a biological complex or process. Such have been the cases for in vitro reconstitution of replication (1), transcription (2,3), translation (4–7) and polyketide/non-ribosomal peptide synthesis (8) in bacteria and translation initiation in eukaryotes (9,10).

In this report, we present a strategy that experimentally reconstructs a biological complex or process (other than bacterial translation) by co-expressing multiple protein components from encoding DNA templates in an in vitro reconstituted protein synthesis system. Use of an in vitro cell-free protein synthesis system allows rapid co-expression of multiple proteins from encoding DNA templates without the potential limitations of heterologous protein expression in living cells, such as biohazard, toxicity and protein folding (11,12). Use of a reconstituted system instead of a cell-extract-based system allows synthesis of proteins in a largely nuclease- and protease-free environment that contains defined components and can be designed to favor protein folding and complex assembly (13,14). This strategy potentially can significantly save the time normally required by conventional in vitro reconstitution methods involving expression and purification of multiple proteins (1–3,8–10).

We apply this strategy to bacterial transcription initiation, the key step for gene regulation in bacteria (15). Transcription initiation in bacteria generally involves recognition of a promoter region by RNA polymerase (RNAP) holoenzyme, comprising RNAP core enzyme (two α subunits, one β subunit, one β′ subunit and one ω subunit) and one of several σ factors (16–19). The primary σ factor (e.g. σ⁷⁰ in Escherichia coli) is responsible for transcription initiation of genes expressed in exponentially growing cells, whereas alternative σ factors are responsible for transcription initiation of genes expressed during stationary phase or in response to specific conditions (e.g. σ³² in E. coli for heat-shock response) (19–21). In vitro reconstitution of E. coli transcription initiation by conventional methods generally involves expression and purification of E. coli RNA polymerase (Ec RNAP) subunits and sigma factors and in vitro assembly of the initiation complex (2,3,22). Here we attempt to simultaneously express the subunits of Ec RNAP and an E. coli sigma factor from encoding DNA templates in a reconstituted protein synthesis system and demonstrate promoter-specific transcription in a single in vitro protein synthesis reaction.

The reconstituted protein synthesis system in this study is derived from PURESYSTEM (4), which comprises purified E. coli ribosomes and bulk tRNAs, purified recombinant E. coli aminoacyl-tRNA synthetases, translation factors and energy regeneration enzymes, and purified recombinant T7 RNA polymerase, which couples transcription from a T7 promoter of a DNA template to protein translation (4,13). Using this reconstituted protein synthesis system, we co-express Ec RNAP holoenzyme subunits from DNA templates under the T7 promoter, and in the same reaction, monitor the activity of the newly synthesized RNAP holoenzyme using a reporter expressed from an E. coli promoter. The reporter, firefly luciferase (Fluc), is encoded on an additional DNA template containing a sigma-specific promoter (Figure 1). Successful synthesis of a functional Ec RNAP holoenzyme leads to transcription of the reporter gene from the sigma-specific promoter and subsequent translation of the reporter protein from the encoding mRNA, resulting in a detectable signal (Figure 1).

Figure 1.

A scheme of coupled expression and detection of E. coli RNA polymerase (Ec RNAP) holoenzyme in a reconstituted protein synthesis system. E. coli RNAP subunits (α, β, β′ and ω) and a sigma factor (σ) are (more ...)

MATERIALS AND METHODS

Reagents

All PCR reactions were performed using Phusion Hot Start High-Fidelity DNA polymerase (New England Biolabs). RNase inhibitor was from New England Biolabs. Primers were ordered from Integrated DNA Technologies (Coralville, IA). Purified Ec RNAP core enzyme and σ⁷⁰-saturated Ec RNAP holoenzyme were purchased from Epicentre Biotechnologies (Madison, WI), bulk tRNA and NTPs from Roche Applied Sciences (Indianapolis, IN) and other chemicals from Sigma-Aldrich (St. Louis, MO). Antibodies against Ec RNAP subunits were kindly provided by Dr Padraig Deighan (Harvard Medical School) and the antibody against Ec σ³² was purchased from Neoclone Biotechnology (Madison, WI).

DNA-template preparation

The plasmid pUCA105T7Fluc was derived from pUC19 containing the gene for Fluc under the control of a T7 promoter (14). The genes for Ec RNAP subunits (α, β, β′ and ω) and E. coli sigma factors (σ⁷⁰ and σ³²) were amplified from E. coli genomic DNA and cloned into pUCA108T7 vector, a derivative of pUCA105T7 vector. The expression of Ec RNAP subunits and sigma factors was under the control of a T7 promoter. The sequences of all cloned genes were verified by DNA sequencing.

For the expression of σ⁷⁰ and σ³², linear DNA templates were prepared by direct amplification from their cloning vector pUCA108T7 σ⁷⁰ and pUCA108T7 σ³², resulting in linear fragments containing the coding regions for σ⁷⁰ and σ³² with an upstream T7 promoter and a downstream T7 terminator. All other linear templates containing non-T7 promoters were prepared by an over-lapping PCR strategy (23) involving two-step PCR reactions that added a promoter region to a gene of interest (Supplementary Figure S2A). More specifically, in the first step (first PCR), the reporter Fluc gene was amplified from pUCA105T7 Fluc to yield a DNA fragment containing the open reading frame (ORF) of Fluc, a 5′ UTR containing the ribosome-binding site and 3′ UTR derived from the vector. The promoter region of dnaKp1 (from −150 to +20) [ … TTGATG(−35) . . 16 nt . . CATTTA(−10) GTAGTCA(+1)] was amplified from E. coli genomic DNA and the promoter region of tac […TTGACA(−35) . . 16 nt . . TATAAT(−10)GTGTGGA(+1)] was amplified from pMalp2x vector (New England Biolabs). The amplification reactions generated a 3′ sequence that overlapped with the 5′ sequence of the reporter DNA fragment, allowing subsequent overlapping PCR reactions (second PCR) to yield the linear DNA templates encoding the reporter gene downstream of different promoters (Supplementary Figure S2A). For best results, DNA fragments from the first PCR step were loaded on 2% agarose gel and then purified by QIAquick gel extraction kit (QIAGEN, Valencia, CA). The purified DNA fragments (~50 ng) were used as the templates for amplification in the second PCR step using the promoter fw primer and 3′ UTR rv primer (Supplementary Figure S2A). DNA templates generated from the second PCR step were purified by QIAquick PCR purification kit (QIAGEN, Valencia, CA) and directly used for protein synthesis reactions.

To generate the linear DNA template for the expression of the wild-type Ec RNAP β subunit, the region containing the β gene, T7 promoter and T7 terminator was directly amplified from the cloning vector pUCA108T7β. To generate linear DNA templates for the expression of single-substitution mutants of the Ec RNAP β subunit, two-step overlapping PCR reactions were performed using mutagenic primer pairs containing the desired mutations (Q513P, Q513L and H526Y) (Supplementary Figure S2B) (23). In the first step (first PCR), pUCA108T7β was used as the template and two PCR reactions were performed for each single-substitution mutation using the upstream fw primer with the reverse mutagenic primer, and the downstream rv primer with the forward mutagenic primer, respectively (Supplementary Figure S2B). For best results, DNA fragments from the first PCR step were loaded on 2% agarose gel and then purified by QIAquick gel extraction kit (QIAGEN, Valencia, CA). The purified DNA fragments (~50 ng) were used as the template for amplification in the second PCR step using the upstream fw primer and downstream rv primer (Supplementary Figure S2B). The resulting DNA fragments, each containing the β subunit gene with a single substitution, T7 promoter and T7 terminator, were used for in vitro protein synthesis reactions. The single substitution mutations were verified by direct DNA sequencing of the PCR-generated DNA fragments.

Reconstituted protein synthesis system

All recombinant proteins including translation factors, aminoacyl-tRNA synthetases, energy-regeneration enzymes and T7 RNA polymerase were kindly provided by Dr Dongxian Yue (New England Biolabs), which were also used for manufacturing PURExpress™, a commercial reconstituted protein synthesis system from New England Biolabs and derived from PURESYSTEM® (BioComber, Tokyo, Japan) (4). Ribosomes were purified according to established protocols (24,25). The reconstituted protein synthesis system was made following essentially the same protocol as Shimizu et al. (13) with a few modifications, including concentrations of aminoacyl-tRNA synthetases, possibly due to differences in specific activities, and potassium glutamate (200 mM instead of 100 mM).

In vitro protein synthesis reactions

The protein synthesis reactions (25 μl) were typically set up by mixing the reconstituted protein synthesis system (20 μl) with RNase inhibitor (2 U), DNA templates and/or purified Ec RNA polymerase (core or holoenzyme) (1.1 pmol in Figure 2A and B or various amounts of the core enzyme in Figure 2C), and then incubated at 37°C for 4 h. The amounts of DNA templates per reaction for all experiments were as follows: 13 nM for linear templates of Fluc (tac-Fluc and dnaKp1-Fluc), 0.07 nM for the linear template of σ⁷⁰ (P_T7-σ⁷⁰) (except in Supplementary Figure S4, in which various amounts were used), 0.25 nM for the linear template of σ³² (P_T7-σ³²), 0.07 nM for the linear templates of the wild-type and mutant β subunits, 0.05 nM for the plasmid templates of RNAP β and β′ subunits, 0.30 nM for the plasmid template of RNAP α subunit, 0.03 nM for the plasmid template of RNAP ω subunit and 4.0 nM for the plasmid template of pUCA105T7Fluc (Supplementary Figure S3). To examine the effect of small-molecule inhibitors, rifampicin (1.2 μM in Figure 4; 0.6 and 1.2 μM in Supplementary Figure S3 and various amounts in Supplementary Figure S5), mupirocin (Mup) (20 μM in Supplementary Figure S3) or chloramphenicol (Cm) (30 μM in Supplementary Figure S3) was added at the beginning of each protein synthesis reaction. The protein concentration of purified Ec RNA polymerase core enzyme was determined by Bradford assay.

Figure 2.

Coupling bacterial transcription to translation in the reconstituted protein synthesis system using purified Ec RNAP. (A) Fluc was expressed under the tac promoter in a linear DNA template in the presence of purified Ec RNAP holoenzyme (column 1, Ec RNAP (more ...)

Figure 4.

Coupled expression and detection of Ec RNAP mutants in the reconstituted protein synthesis system. The Fluc activity was measured in protein synthesis reactions containing the linear DNA templates of dnaKp1-Fluc and PT7-σ³², separate plasmid templates (more ...)

Western blot analyses

Aliquots were taken from protein synthesis reactions after incubation at 37°C for 4 h and loaded on 10–20% Tris–glycine SDS–PAGE gels (Invitrogen, Carlsbad, CA). The gels were blotted for analyses with antibodies against Ec RNAP subunits and σ³² using Phtotope-HRP Western Blot Detection System (Cell Signaling Technology, Beverly, MA).

Luciferase assay

The activity of the Fluc reporter was assayed using the Luciferase Assay System (Promega, Madison, WI) in a microplate luminometer (Centro LB 640, Berthold Technologies, Oak Ridge, TN) according to manufacturers’ instructions. Protein synthesis reactions were diluted 10-fold in 1× cell culture lysis reagent (Promega, Madison, WI) containing 1 mg/ml BSA. Aliquots (5 μl) were added in triplicate to a microplate for the luciferase assay in the luminometer.

RESULTS

Coupling Ec RNAP transcription to translation using purified enzymes

The reconstituted protein synthesis system contains T7 RNA polymerase to couple transcription to translation from a double-stranded DNA template containing a T7 promoter (4). A previous study has showed that the amount of synthesized protein in PURESYSTEM is largely correlated to the amount of the encoding mRNA (within a certain concentration range) transcribed by T7 RNA polymerase (26). We have previously demonstrated the detection of the activity of Fluc synthesized from a DNA template under a T7 promoter in PURESYSTEM (14). In this study, we first tested whether the synthesis and activity of Fluc could be correlated to the sigma-factor-dependent transcription by purified Ec RNAP in the reconstituted protein synthesis system. Purified recombinant σ⁷⁰-saturated Ec RNAP holoenzyme or Ec RNAP core enzyme was added to the protein synthesis reaction containing a linear DNA template (tac-Fluc) encoding Fluc under the control of a tac promoter (27). As shown in Figure 2A, a significant increase in the activity of Fluc was observed in the presence of σ⁷⁰-saturated Ec RNAP holoenzyme (Figure 2A, Ec RNAP holo) compared to Ec RNAP core enzyme (Figure 2A, Ec RNAP core). The data suggest that the σ⁷⁰-dependent transcription initiation from the tac promoter and the transcription of the full-length Fluc gene by Ec RNAP resulted in the translation of active Fluc from the encoding mRNA. No significant Fluc activity was detected when purified Ec RNAP was not added to the protein synthesis reaction (Figure 2A, no RNAP), suggesting the reconstituted protein synthesis system used in this study was largely free of contaminating Ec RNAP activity (also see data in Figure 3C). As another control, no Fluc activity was detected in the absence of the Fluc template even when purified Ec RNAP was added in the protein synthesis reaction (Figure 2A, no DNA template).

Figure 3.

Coupled expression and detection of Ec RNAP holoenzymes in the reconstituted protein synthesis system using the encoding DNA templates. (A) The Fluc activity was measured in the protein synthesis reactions containing the linear DNA templates of P_T7-σ (more ...)

These data establish preliminary evidence that transcription (including transcription initiation) by Ec RNAP was compatible with, and could be coupled to, translation in the reconstituted protein synthesis system, and the activity of Fluc was correlated to the in vitro transcription activity of Ec RNAP.

Synthesis of functional E. coli sigma factors and demonstration of promoter-specific expression

We next tested whether the reconstituted protein synthesis system could be used to produce E. coli sigma factors from the encoding DNA templates to allow promoter-specific transcription in the presence of purified Ec RNA core enzyme.

The protein synthesis reactions contained linear DNA templates encoding Fluc under an E. coli promoter, tac (27) or dnaKp1 [an E. coli σ³²-specific promoter (28)] (tac-Fluc or dnaKp1-Fluc), an E. coli sigma factor σ⁷⁰ or σ³² under the T7 promoter (P_T7-σ⁷⁰ or P_T7-σ³²) and the purified Ec RNAP core enzyme (the same amount as in Figure 2A, Ec RNAP core). A significant increase in the activity of Fluc was observed from the tac-Fluc template in the presence of P_T7-σ⁷⁰ (Figure 2B, tac-Fluc, shaded column) compared to in the absence of P_T7-σ⁷⁰ (Figure 2A, Ec RNAP core). Though the Fluc activity in the presence of P_T7-σ⁷⁰ was still lower than that of σ⁷⁰-saturated Ec RNAP holoenzyme (Figure 2A, Ec RNAP holo), the data suggest that a functional σ⁷⁰ was produced from P_T7-σ⁷⁰ in the protein synthesis reaction, forming a holoenzyme with the purified Ec RNAP core enzyme to allow transcription initiation from tac-Fluc. As further evidence, in the presence of σ⁷⁰, a higher Fluc activity was observed from the tac-Fluc template than from the dnaKp1-Fluc template (Figure 2B, shaded columns). In comparison, in the presence of σ³², a higher Fluc activity was observed from the dnaKp1-Fluc template than from the tac-Fluc template (Figure 2B, white columns). The data are consistent with the cellular roles of σ⁷⁰ as the housekeeping sigma factor and σ³² as the sigma factor for heat-shock response (18,29), further supporting that functional E. coli sigma factors were synthesized and assembled into Ec RNAP holoenzymes capable of initiating transcription from specific E. coli promoters. In spite of the σ⁷⁰ consensus sequence in the tac promoter, a higher Fluc activity was observed from the dnaKp1 promoter in the presence of σ³² than that from the tac promoter in the presence of σ⁷⁰. One possible reason was the effect of the concentration of potassium glutamate in the reconstituted protein synthesis system on in vitro transcription (19,30). High concentrations of potassium glutamate have been shown to inhibit in vitro transcription by σ⁷⁰ (30), whereas lowering the concentration of potassium glutamate (from 200 to 100 mM) decreased the Fluc activity from the σ³²/dnaKp1-Fluc templates by ~50% (data not shown). Another possible factor was the folding of the synthesized σ⁷⁰, which might result in a lower specific activity than that of σ³².

Using the fixed amounts of the P_T7-σ³² and dnaKp1-Fluc templates, the Fluc activity was determined in the protein synthesis reactions containing increasing amounts of purified Ec RNAP core enzyme. As shown in Figure 2C, the Fluc activity increased proportionally to the amount of Ec RNAP core enzyme. The data provide a basis for the correlation between the Fluc activity and the amount of active Ec RNAP core enzyme in the reconstituted protein synthesis system. If the detection limit is defined as a signal-to-noise ratio (the ratio of the two Fluc activities in the presence and absence of the core enzyme, respectively) = 2, the data in Figure 2C suggest that as few as ~0.025 pmol (~1 nM) of Ec RNAP core enzyme (presumably also RNAP holoenzyme) can be detected in the reconstituted protein synthesis reaction.

Coupled expression and detection of Ec RNAP holoenzymes using the encoding DNA templates

To demonstrate the synthesis of the holoenzymes, the protein synthesis reactions contained the P_T7-σ³² and dnaKp1-Fluc templates and separate plasmid templates encoding all four (P_T7-αββ′ω) or three out of four subunits of Ec RNAP (P_T7-ββ′ω, P_T7-αβ′ω, P_T7-αβω or P_T7-αββ′). No purified Ec RNAP core enzyme was added in the protein synthesis reactions. Synthesis of all four Ec RNA subunits and σ³² from their DNA templates and assembly of a functional Ec RNAP holoenzyme would lead to transcription of Fluc from the σ³²-specific dnaKp1 promoter and detection of the Fluc activity.

As shown in Figure 3A, a significant Fluc activity was observed in the presence of the templates encoding all four or the α, β and β′ subunits of Ec RNAP (P_T7-αββ′ω and P_T7-αββ′). In the absence of one of the templates for α, β and β′, the Fluc activity was reduced to the background levels (Figure 3A, P_T7-ββ′ω, P_T7-αβ′ω and P_T7-αβω). The data suggest that a functional Ec RNAP core enzyme consisting of at least three subunits (α, β and β′) was produced in the reconstituted protein synthesis system and all three subunits (α, β and β′) were required for the assembly of a functional Ec RNAP core enzyme. The expression of the α, β and β′ subunits and σ³² was further confirmed by western blot analyses using antibodies against each of the subunits and the sigma factor (Supplementary Figure S1). The similar Fluc activities observed in the presence and absence of the P_T7-ω template (Figure 3A, P_T7-αββ′ω and P_T7-αββ′) suggest that the ω subunit was not required for the in vitro transcription activity of the synthesized Ec RNAP core enzyme, consistent with previous studies (31,32). However, since the expression of a functional ω subunit from its template was not confirmed in this study, we cannot rule out the possibility of the contaminating ω subunit in the reconstituted protein synthesis system. More analyses are needed to distinguish these scenarios.

The synthesis of a functional Ec RNAP core enzyme was further tested in the protein synthesis reactions containing the templates encoding all four subunits, the sigma factor template (P_T7-σ⁷⁰ or P_T7-σ³²) and the Fluc template (tac-Fluc or dnaKp1-Fluc). As shown in Figure 3B, in the presence of P_T7-σ⁷⁰, a significantly higher Fluc activity was observed from the tac promoter than the dnaKp1 promoter (Figure 3B, shaded columns). In contrast, in the presence of P_T7-σ³², a much higher Fluc activity was observed from the dnaKp1 promoter than the tac promoter (Figure 3B, white columns). The data are consistent with the known functions of Ec RNAP core enzyme during transcription initiation at different promoters (18,28), further supporting that a functional Ec RNAP core enzyme was indeed synthesized to form Ec RNAP holoenzymes with the sigma factors, capable of initiating transcription from specific E. coli promoters.

A series of control experiments were performed to further quantify the background-level expression of Fluc in the absence of sigma-factor templates and/or RNAP subunit templates. In the first set of controls (Figure 3C, columns 1–3), the Fluc templates (tac-Fluc and dnaKp1-Fluc) were absent in the protein synthesis reactions. No Fluc activity was detected, indicating that there was no contaminating Fluc gene in the DNA templates of Ec RNAP subunits and sigma factors. In the second set of controls (Figure 3C, columns 4–6), the Fluc activity was measured from two Fluc templates (tac-Fluc and dnaKp1-Fluc) in the absence of other DNA templates. The Fluc activity observed for the dnaKp1-Fluc template (columns 5) was reduced significantly in the absence of T7 RNA polymerase (T7 RNAP, column 4), suggesting that T7 RNA polymerase present in the reconstituted protein synthesis system was partially responsible for the background transcription activity from the Fluc templates even though the expression of Fluc was under the control of E. coli promoters. Promoter-independent transcription by T7 RNA polymerase has been previously reported (33). Though no Ec RNAP subunits were detected in the absence of their DNA templates by the western blot analysis (Supplementary Figure S1, lane 1), we cannot rule out that a low level of endogenous RNAP from E. coli was present in the reconstituted protein synthesis system and was responsible for some of the background transcription activity from the Fluc templates. The notion of contaminating Ec RNAP activity in the reconstituted protein synthesis system was also suggested by the next set of controls that included the templates for the sigma factors but not for Ec RNAP subunits (Figure 3C, columns 7–10). In the presence of the σ³² template (P_T7-σ³²), a higher Fluc activity was observed from the dnaKp1 promoter (column 7) than the tac promoter (column 8). Similarly, in the presence of the σ⁷⁰ template (P_T7-σ⁷⁰), a higher Fluc activity was observed from the tac promoter (column 10) than the dnaKp1 promoter (column 9). As the last set of controls, the Fluc activity was measured in the absence of the templates for the sigma factors (Figure 3C, columns 11 and 12). Addition of the templates of Ec RNAP subunits did not result in significant further increase in the Fluc activity (Figure 3C, compare columns 11 and 12 with columns 5 and 6). Only when the templates for the sigma factors and Ec RNAP subunits were all present, a significant increase in the Fluc activity was observed from both Fluc templates (Figure 3B, note the Fluc activity at a different scale from that in Figure 3C). The signal-to-noise ratio (defined as the ratio of the two Fluc activities in the presence and absence of the RNAP subunit genes, respectively) is ~20 for both σ⁷⁰/tac-Fluc and σ³²/dnaKp1Fluc templates. A higher signal-to-noise ratio (>100) was obtained for Ec σ²⁸ when Fluc was expressed from a σ²⁸-specific promoter, largely due to a very low Fluc activity (noise) in the absence of the RNAP subunit genes (data not shown).

Taken together, above data suggest that Ec RNAP subunits (at least α, β and β′) were indeed produced from encoding DNA templates in the protein synthesis reactions and subsequently assembled into a functional Ec RNAP core enzyme capable of catalyzing sigma-factor-dependent, promoter-specific transcription initiation.

Rapid generation and functional detection of Ec RNAP mutants using PCR-generated DNA templates

Coupled expression and detection of Ec RNAP holoenzyme in the reconstituted protein synthesis system presents a potential platform for rapid in vitro analyses of bacterial transcription initiation and RNAP holoenzymes. Since DNA templates can be used for the expression of Ec RNAP subunits, the method would be particularly useful for characterization of Ec RNAP mutants. Previous studies have identified Q513 and H526 of the β subunit of Ec RNAP as two of key residues for the binding of the small-molecule inhibitor rifamycin (34–36). Single substitutions of either residue have been found to confer rifamycin-resistance both in vitro and in vivo (34,36). We made three such single substitutions in the β subunit of Ec RNAP (i.e. Q513P, Q513L and H526Y) (34) using an overlapping PCR strategy (23) (Supplementary Figure S2B). The resulting linear DNA templates, encoding the β mutants under a T7 promoter [P_T7-β(Q513P), P_T7-β(Q513L) and P_T7-β(H526L)], were directly used for the protein synthesis reactions in which other Ec RNAP subunits were expressed from separate plasmid templates (Figure 4, P_T7-αβ′ω). As a control, the wild-type β subunit was also expressed from a linear DNA template [P_T7-β (WT)] generated by direct amplification from its plasmid template. For comparison, the wild-type RNAP core enzyme was expressed from all-plasmid templates (Figure 4, P_T7-αββ′ω). To monitor the activity of the synthesized Ec RNAP, the P_T7-σ³² and dnaKp1-Fluc templates were used.

As shown in Figure 4, the β subunit expressed from the linear template [P_T7-β (WT), column 3] or the plasmid template (as in P_T7-αββ′ω, column 1) resulted in a similar Fluc activity, suggesting that both templates produced functional wild-type RNAP core enzyme. Addition of rifampicin (one of rifamycins) during the expression of the wild-type RNAP subunits significantly reduced the Fluc activity (Figure 4, column 4), to a level similar to when the template for the β subunit [P_T7-β (WT)] was absent in the protein synthesis reaction (Figure 4, column 2). Rifampicin at similar concentrations did not inhibit protein translation in the reconstituted protein synthesis system (Supplementary Figure S3). The data suggest that rifampicin inhibited the activity of the wild-type Ec RNAP core enzyme synthesized in the protein synthesis reaction, consistent with previous reports (36).

In the absence of rifampicin, the expression of the single-substitution mutants of the β subunit resulted in considerable Fluc activity compared to the expression of the wild-type β subunit [β (WT)] (Figure 4, columns 3, 5, 7 and 9), suggesting that the synthesized RNAP mutants were transcriptionally active. In contrast to β (WT) (Figure 4, column 4), all three single-substitution β mutants resulted in a significant Fluc activity in the presence of rifampicin (Figure 4, columns 6, 8 and 10), suggesting that the synthesized RNAP mutants were also rifampicin-resistant. The IC₅₀s for all three β mutants, defined as the concentrations of rifampicin necessary for the inhibition of the Fluc activity by 50%, were >10 μM, whereas the IC₅₀ for β (WT) was ~0.2 μM (Supplementary Figure S5). The data are consistent with previous in vivo studies and in vitro data using purified mutant enzymes (34–36).

DISCUSSION

In this study, we demonstrated in vitro reconstruction of basic E. coli transcription initiation from DNA templates in a reconstituted protein synthesis system. By adding purified Ec RNAP holoenzyme, we first established the reporter system and the reaction condition for coupled (Ec RNAP-initiated) transcription and translation. By adding purified Ec RNAP core enzyme, we then demonstrated the synthesis of the functional E. coli sigma factors from their encoding DNA templates. By using the DNA templates encoding Ec RNAP subunits and sigma factors, we finally demonstrated the synthesis and assembly of the functional E. coli holoenzymes. Successful synthesis of Ec RNAP subunits and sigma factors, and functional assembly of Ec RNAP holoenzymes for transcription initiation were supported by the sigma-factor-dependent, promoter-specific transcription of the reporter gene (Figures 2 and and3),3), the rifampicin-sensitive activity due to the expression of the wild-type β subunit, the rifampicin-resistant activity due to the expression of the single-substitution β mutants (Figure 4, Supplementary Figure S5) and western blot analyses of the expression of Ec RNAP subunits (Supplementary Figure S1).

By reconstructing E. coli transcription initiation, we also demonstrated the production of multiple proteins from their encoding DNA templates in the reconstituted protein synthesis system. In the case of Ec RNAP holoenzyme, at least five proteins (α, β, β′, σ and Fluc) with molecular weights ranging from 32 kD to 155 kDa were produced from separate DNA templates in a single protein synthesis reaction. The synthesis of a functional Ec RNAP ω subunit was not confirmed in this study since no significant change in the Fluc activity was observed in the absence of the ω template (P_T7-ω) (Figure 3A). PCR-generated linear templates were used for the synthesis of Fluc and sigma factors, whereas circular plasmid templates were used for the synthesis of four Ec RNAP subunits (Figure 3). One of the plasmid templates for Ec RNAP subunits can be replaced by a separate PCR-generated linear template without significantly affecting the activity of the synthesized RNAP holoenzyme (Figure 4, column 3). In fact, all plasmid templates for Ec RNAP subunits can be replaced by PCR-generated linear templates without significantly affecting the activity of the synthesized RNAP holoenzyme (data not shown). Use of PCR-generated linear templates allowed rapid generation and analysis of mutants without cloning steps (Supplementary Figure S2).

Since the same protein translation machinery was responsible for the production of multiple proteins in the reconstituted protein synthesis system, it was necessary to balance the amounts of proteins synthesized for the assembly of the protein complex (e.g. RNAP holoenzyme) and for the detection of the reporter activity (e.g. Fluc activity). In protein synthesis reactions, the amount of the template for Fluc was kept higher than the amounts of the templates for sigma factors and RNAP subunits (see ‘Materials and Methods’ section), due to the consideration that the expression of Fluc was under relatively weak E. coli promoters compared to the T7 promoter (37), which was used for the expression of sigma factors and RNAP subunits. Increasing the amount of the template for the sigma factor resulted in a decrease in the Fluc activity (Supplementary Figure S4). One limitation may be the protein synthesis capacity of the reconstituted protein synthesis system. The optimal ratio for the relative amounts of the templates encoding sigma factors and RNAP subunits was not determined in this study. A systematic optimization may be necessary to further increase the amount and specific activity of the synthesized RNAP holoenzyme as well as the signal generated by the Fluc activity.

In vitro reconstruction of E. coli transcription initiation demonstrated in this study can contribute to the study of not only known RNAP from model bacteria but also uncharacterized RNAP from other bacteria. Such approach has many potential advantages.

First, each RNAP subunit or sigma factor is encoded on a separate plasmid or linear DNA template, making cloning and site-specific mutagenesis a simple task and comprehensive mutational analyses a possibility. Using such strategy, it is possible to generate a large number of substitutions in the genes of RNAP subunits and sigma factors within a reasonable period of time, allowing high-througput initial analyses of protein–protein and protein–small-molecule interactions. Using the conventional reconstitution methods, on the other hand, such tasks would be prohibitively laborious and time-consuming. Furthermore, RNAP or RNAP mutants that are toxic to the host cell due to interference with critical cellular functions or rapidly degraded due to low stability can not be produced in vivo.

Second, use of an additional DNA template encoding a reporter protein under the control of a sigma-factor-specific promoter allows immediate detection of an active RNAP holoenzyme, eliminating the need for purification of RNAP and a separate in vitro transcription step, and saving, possibly by orders-of-magnitude, the time normally taken by the conventional reconstitution methods. Due to the presence of endogenous Ec RNAP and transcription factors, this coupled synthesis and detection strategy may not work for the expression of other bacterial RNAP in E. coli or in the E. coli-extract-based cell-free systems. Based on western blot analyses, the endogenous Ec RNAP and sigma factors (and possibly transcription factors) were not detectable in our reconstituted protein synthesis system (Supplementary Figure S1, lane 1). Therefore, the reconstituted system may provide more consistent and cleaner preparation of RNAP for functional studies, free of host binding proteins or host-hybrid enzymes (22). The drawbacks of in vitro synthesis of RNAP and sigma factors are that the amounts of synthesized proteins are low and not easily determined. Therefore, for detailed (follow-up) mechanistic studies, the conventional reconstituted methods may be preferred.

Third, we speculate that in vitro protein synthesis in the background of a low level of nucleases and proteases may benefit the folding and complex assembly of multi-component enzymes. The rate of the nascent chain elongation on ribosomes in a cell-free system is estimated to be ~2 residues/s (in contrast to ~20 residues/s in growing E. coli cells) (38), which may allow more time for proper folding and/or co-folding with other proteins in the reconstituted protein synthesis system than in vivo. This benefit is probably lost in the cell-extract-based systems as incomplete folded proteins may be degraded by proteases and unprotected mRNA may be degraded by nucleases before the productive assembly of a protein complex can be achieved.

Lastly, the in vitro synthesis and function of RNAP are correlated to the activity of a luminescent reporter protein commonly used in high-throughput assays. Therefore, the system is instantly ready for microplate-format high-throughput screening of small-molecule inhibitors of RNAP, a proven target for antibiotics (39). Since potential small-molecule inhibitors can be added during the synthesis, assembly and catalysis (transcription initiation) of RNAP, we speculate that our approach, resembling the natural processes inside the cell, can potentially lead to novel inhibitors that target the sites of RNAP not yet found by conventional in vitro screening methods in which purified and preassembled RNAP are normally used (40).

Reconstruction of E. coli transcription initiation in the reconstituted protein synthesis system may provide an initial step towards experimental reconstruction of more complex scenarios of bacterial transcription from genetic materials (DNA templates). For instance, additional sigma factors may be synthesized from their encoding DNA templates for functional analyses of multiple promoter regions, which can be linked to one or multiple reporter genes. Repressors and activators may also be synthesized in the same protein synthesis reaction for reconstruction of certain regulatory pathways in bacterial transcription. The ability of using multiple and PCR-generated DNA templates in the reconstituted protein synthesis system may allow rapid analyses of a large number of genes and promoters involved in bacterial transcription and its regulation. Future experiments will examine if low transcription by weak promoters or transcription activation/repression can be detected by, and correlated to, the activity of Fluc or other reporters in the reconstituted protein synthesis system. Another unanswered question is the maximal number of proteins that can be simultaneously synthesized from their DNA templates and detected as the result of their functions in the reconstituted protein synthesis system. Efforts to further enhance the synthesis and folding capacity of the reconstituted system are underway.

One can also envision experimental reconstruction of bacterial replication in the reconstituted protein synthesis system using DNA templates containing an origin of replication and genes encoding DNA polymerases and other components. In fact, our preliminary studies have suggested that coupled expression and detection of an E. coli DNA polymerase is possible in the reconstituted proteins synthesis system and that the DNA-template-directed polymerization reaction is compatible with the condition of the protein synthesis reaction (S. Chong, unpublished). A reconstituted system capable of demonstrating replication, transcription and translation would facilitate in vitro study and intervention of three polymerization processes that are essential to all life.

In summary, reconstruction of E. coli transcription initiation in this study illustrates a general strategy for in vitro experimental reconstruction of a multi-component biological complex or process. The strategy couples expression of multiple gene products with detection of the resulting biological activities in a reconstituted protein synthesis system and allows rapid in vitro reconstruction of a functional biological complex or process from DNA templates instead of purified components. Since protein synthesis, complex assembly and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles in vivo biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by the conventional reconstitution methods and provide an open and designable platform for in vitro study and intervention of complex biological processes. The key for successful application of such strategy is the ability of the reconstituted protein synthesis system to produce multiple gene products in functional forms. The reconstituted protein synthesis system may be further modified by addition of new components, and/or optimization of existing components, in order to increase the capacity of protein synthesis and facilitate folding, assembly and detection of multi-gene products. Here we propose to name such a reconstituted system an ‘Expressome’. Current reconstituted protein synthesis systems have largely been used as superior cell-free systems over cell-extract-based systems for synthesis of single proteins (11–14,41), incorporation of unnatural amino acids (4,42,43), study of nascent chain folding (44,45), in vitro protein evolution (41,46,47) and synthetic-cell applications involving few genes (48,49). In comparison, an Expressome contains, in addition to all the components of a reconstituted protein synthesis system, DNA templates designed to encode a set of genes involved in a particular biological complex or process and (if necessary) one or several reporter genes that facilitate detection. In other words, an Expressome is an in vitro reconstruction of a biological complex or process beyond protein translation and with higher complexity. A potential advantage of an Expressome would be to enable a ‘cell-free genetic approach’ to study biological functions, allowing genetic alterations to be rapidly generated and detected in vitro. Since E. coli has been extensively studied with widely available genetic tools and databases (50), the approach would be particularly valuable for the study of bacteria and other organisms with known genome sequences but for which no or limited genetic tools are available.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

Funding for open access charge: New England Biolabs Inc.

Conflict of interest statement. None declared.

Supplementary Material

[Supplementary Data]

Click here to view.

ACKNOWLEDGEMENTS

We are grateful to Dr Christopher Noren, Dr Elisabeth Raleigh and Dr William Jack for critical reading of the manuscript; Dr Dongxian Yue for providing recombinant proteins for making the reconstituted protein synthesis system; Dr Yoshihiro Shimizu and Prof Takuya Ueda for stimulating discussion; Dr Richard Ebright, Dr Padraig Deighan and Dr Ann Hochschild for insightful discussion and providing the RNAP plasmid and antibodies; and Dr Don Comb for encouragement.

REFERENCES

1. Yuzhakov A, Turner J, O'Donnell M. Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication. Cell. 1996;86:877–886. [PubMed]

2. Fujita N, Ishihama A. Reconstitution of RNA polymerase. Methods Enzymol. 1996;273:121–130. [PubMed]

3. Tang H, Severinov K, Goldfarb A, Ebright RH. Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase. Proc. Natl Acad. Sci. USA. 1995;92:4902–4906. [PubMed]

4. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 2001;19:751–755. [PubMed]

5. Kung HF, Redfield B, Treadwell BV, Eskin B, Spears C, Weissbach H. DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors. J. Biol. Chem. 1977;252:6889–6894. [PubMed]

6. Ganoza MC, Cunningham C, Green RM. Isolation and point of action of a factor from Escherichia coli required to reconstruct translation. Proc. Natl Acad. Sci. USA. 1985;82:1648–1652. [PubMed]

7. Pavlov MY, Ehrenberg M. Rate of translation of natural mRNAs in an optimized in vitro system. Arch. Biochem. Biophys. 1996;328:9–16. [PubMed]

8. Sattely ES, Fischbach MA, Walsh CT. Total biosynthesis: in vitro reconstitution of polyketide and nonribosomal peptide pathways. Nat. Prod. Rep. 2008;25:757–793. [PubMed]

9. Kolupaeva VG, de Breyne S, Pestova TV, Hellen CU. In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry. Methods Enzymol. 2007;430:409–439. [PubMed]

10. Acker MG, Kolitz SE, Mitchell SF, Nanda JS, Lorsch JR. Reconstitution of yeast translation initiation. Methods Enzymol. 2007;430:111–145. [PubMed]

11. Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 2005;23:150–156. [PubMed]

12. Jermutus L, Ryabova LA, Pluckthun A. Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 1998;9:534–548. [PubMed]

13. Shimizu Y, Kanamori T, Ueda T. Protein synthesis by pure translation systems. Methods. 2005;36:299–304. [PubMed]

14. Hillebrecht JR, Chong S. A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based. BMC Biotechnol. 2008;8:58. [PMC free article] [PubMed]

15. Young BA, Gruber TM, Gross CA. Views of transcription initiation. Cell. 2002;109:417–420. [PubMed]

16. Campbell EA, Westblade LF, Darst SA. Regulation of bacterial RNA polymerase sigma factor activity: a structural perspective. Curr. Opin. Microbiol. 2008;11:121–127. [PMC free article] [PubMed]

17. Murakami KS, Darst SA. Bacterial RNA polymerases: the wholo story. Curr. Opin. Struct. Biol. 2003;13:31–39. [PubMed]

18. Gruber TM, Gross CA. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 2003;57:441–466. [PubMed]

19. Ishihama A. Functional modulation of Escherichia coli RNA polymerase. Annu. Rev. Microbiol. 2000;54:499–518. [PubMed]

20. Guisbert E, Yura T, Rhodius VA, Gross CA. Convergence of molecular, modeling, and systems approaches for an understanding of the Escherichia coli heat shock response. Microbiol. Mol. Biol. Rev. 2008;72:545–554. [PMC free article] [PubMed]

21. Alba BM, Gross CA. Regulation of the Escherichia coli sigma-dependent envelope stress response. Mol. Microbiol. 2004;52:613–619. [PubMed]

22. Kuznedelov K, Severinov K. Recombinant bacterial RNA polymerase: preparation and applications. Methods. 2009;47:44–52. [PubMed]

23. Pogulis RJ, Vallejo AN, Pease LR. In vitro recombination and mutagenesis by overlap extension PCR. Methods Mol. Biol. 1996;57:167–176. [PubMed]

24. Ohashi H, Shimizu Y, Ying BW, Ueda T. Efficient protein selection based on ribosome display system with purified components. Biochem. Biophys. Res. Commun. 2007;352:270–276. [PubMed]

25. Korostelev A, Trakhanov S, Laurberg M, Noller HF. Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell. 2006;126:1065–1077. [PubMed]

26. Maki K, Uno K, Morita T, Aiba H. RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA. Proc. Natl Acad. Sci. USA. 2008;105:10332–10337. [PubMed]

27. de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl Acad. Sci. USA. 1983;80:21–25. [PubMed]

28. Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA. Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress. Genes Dev. 2006;20:1776–1789. [PubMed]

29. Gross CA, Chan C, Dombroski A, Gruber T, Sharp M, Tupy J, Young B. The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb. Symp. Quant. Biol. 1998;63:141–155. [PubMed]

30. Kundu TK, Kusano S, Ishihama A. Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes. J. Bacteriol. 1997;179:4264–4269. [PMC free article] [PubMed]

31. Vrentas CE, Gaal T, Ross W, Ebright RH, Gourse RL. Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA. Genes Dev. 2005;19:2378–2387. [PubMed]

32. Mathew R, Chatterji D. The evolving story of the omega subunit of bacterial RNA polymerase. Trends Microbiol. 2006;14:450–455. [PubMed]

33. Zaher HS, Unrau PJ. T7 RNA polymerase mediates fast promoter-independent extension of unstable nucleic acid complexes. Biochemistry. 2004;43:7873–7880. [PubMed]

34. Jin DJ, Gross CA. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J. Mol. Biol. 1988;202:45–58. [PubMed]

35. Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst SA. Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell. 2001;104:901–912. [PubMed]

36. Floss HG, Yu TW. Rifamycin-mode of action, resistance, and biosynthesis. Chem. Rev. 2005;105:621–632. [PubMed]

37. Tabor S. Expression using the T7 RNA polymerase/promoter system. Curr. Protoc. Mol. Biol. 2001 Chapter 16, Unit 1612. [PubMed]

38. Underwood KA, Swartz JR, Puglisi JD. Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis. Biotechnol. Bioeng. 2005;91:425–435. [PubMed]

39. Chopra I. Bacterial RNA polymerase: a promising target for the discovery of new antimicrobial agents. Curr. Opin. Investig. Drugs. 2007;8:600–607. [PubMed]

40. Kozlov M, Bergendahl V, Burgess R, Goldfarb A, Mustaev A. Homogeneous fluorescent assay for RNA polymerase. Anal. Biochem. 2005;342:206–213. [PubMed]

41. Shimizu Y, Kuruma Y, Ying BW, Umekage S, Ueda T. Cell-free translation systems for protein engineering. FEBS J. 2006;273:4133–4140. [PubMed]

42. Josephson K, Hartman MC, Szostak JW. Ribosomal synthesis of unnatural peptides. J. Am. Chem. Soc. 2005;127:11727–11735. [PubMed]

43. Tan Z, Blacklow SC, Cornish VW, Forster AC. De novo genetic codes and pure translation display. Methods. 2005;36:279–290. [PubMed]

44. Kaiser CM, Chang HC, Agashe VR, Lakshmipathy SK, Etchells SA, Hayer-Hartl M, Hartl FU, Barral JM. Real-time observation of trigger factor function on translating ribosomes. Nature. 2006;444:455–460. [PubMed]

45. Ying BW, Taguchi H, Ueda T. Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding. J. Biol. Chem. 2006;281:21813–21819. [PubMed]

46. Zheng Y, Roberts RJ. Selection of restriction endonucleases using artificial cells. Nucleic Acids Res. 2007;35:e83. [PMC free article] [PubMed]

47. Villemagne D, Jackson R, Douthwaite JA. Highly efficient ribosome display selection by use of purified components for in vitro translation. J. Immunol. Methods. 2006;313:140–148. [PubMed]

48. Sunami T, Matsuura T, Suzuki H, Yomo T. Synthesis of functional proteins within liposomes. Methods Mol. Biol. 2010;607:243–256. [PubMed]

49. Chiarabelli C, Stano P, Luisi PL. Chemical approaches to synthetic biology. Curr. Opin. Biotechnol. 2009;20:492–497. [PubMed]

50. Keseler IM, Bonavides-Martinez C, Collado-Vides J, Gama-Castro S, Gunsalus RP, Johnson DA, Krummenacker M, Nolan LM, Paley S, Paulsen IT, et al. EcoCyc: a comprehensive view of Escherichia coli biology. Nucleic Acids Res. 2009;37:D464–D470. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of
Oxford University Press