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

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.

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 encoded in separate linear or plasmid DNA templates under the control of a T7 promoter (P_T7). A reporter, firefly luciferase (Fluc), is encoded in an additional linear DNA template under the control of an E. coli promoter (P_Ec). The synthesized Ec RNAP subunits are assembled into RNAP core enzyme (Ec RNAP core) and subsequently form the holoenzyme with the sigma factor (σ) during transcription initiation. rbs: ribosome-binding site; AUG: start codon.

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 holo) or Ec RNAP core enzyme (column 2, Ec RNAP core). As controls, the Fluc activity was measured in the absence of Ec RNAP holoenzyme (but in the presence of the Fluc template) (column 3, no RNAP) and in the absence of the Fluc template (but in the presence of purified Ec RNAP holoenzyme) (column 4, no DNA template). (B) Fluc was expressed under two different promoters in linear DNA templates (tac-Fluc and dnaKp1-Fluc) in the presence of purified Ec RNAP core enzyme. The Fluc activity was measured in the presence of the template for σ⁷⁰ (P_T7-σ⁷⁰, shaded columns) or σ³² (P_T7-σ³², white columns). (C) The Fluc activity was measured from the templates of dnaKp1-Fluc and P_T7-σ³² in the presence of various amounts (pmol per reaction) of purified Ec RNAP core enzyme. All data were obtained from at least two independent protein synthesis reactions.

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-σ³² and dnaKp1-Fluc and the 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′). (B) The Fluc activity was measured in the protein synthesis reactions containing the linear DNA template encoding Fluc under tac or dnaKp1 promoter (tac-Fluc or dnaKp1-Fluc), the linear DNA template encoding σ⁷⁰ or σ³² (P_T7-σ⁷⁰, shaded columns; P_T7-σ³², white columns) and the separate plasmid templates encoding all four Ec RNAP subunits (P_T7-αββ′ω). (C) Control experiments to determine the background levels of the Fluc activity in the reconstituted protein synthesis system in the presence or absence of various DNA templates. The reconstituted protein synthesis system containing T7 RNA polymerase (T7 RNAP) were used for all experiments except in column 4 in which T7 RNAP was absent. All data were obtained from at least three independent protein synthesis reactions.

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 encoding all four or three wild-type Ec RNAP subunits (P_T7-αββ′ω or P_T7-αβ′ω) and the PCR-generated linear template for the wild-type β subunit [P_T7-β (WT)] or the single-substitution β subunit mutant [P_T7-β (Q513P), P_T7-β (Q513L) or P_T7-β (H526Y)] in the absence or presence of rifampicin (1.2 μM). The relative Fluc activity was presented with the activity from the template of P_T7-αββ′ω set as 100. All data were obtained from at least two independent protein synthesis reactions.