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 ( and ), 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 (, 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 (PT7
-ω) (A). 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 (). 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 (, 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
), 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
), incorporation of unnatural amino acids (4
), study of nascent chain folding (44
), in vitro
protein evolution (41
) and synthetic-cell applications involving few genes (48
). 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.