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
Received March 3, 2010; Revised April 23, 2010; Accepted April 26, 2010
In vitro reconstitution of a biological complex or
process normally involves assembly of multiple in-
dividually 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 synthe-
sis system. The strategy potentially allows experi-
biological complex or process using only DNA tem-
plates 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
demonstrated sigma-factor-dependent, promoter-
specific transcription initiation. Since protein syn-
thesis, complex formation and enzyme catalysis
occur in the same in vitro reaction mixture, this re-
construction process resembles natural biosynthet-
ic 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
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), trans-
lation (4–7) and polyketide/non-ribosomal peptide synthe-
sis (8) in bacteria and translation initiation in eukaryotes
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 heterol-
ogous 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 syn-
thesis 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 reconsti-
tution methods involving expression and purification of
multiple proteins (1–3,8–10).
We apply this strategy to bacterial transcription initi-
ation, the key step for gene regulation in bacteria (15).
Transcription initiation in bacteria generally involves rec-
ognition of a promoter region by RNA polymerase
(RNAP) holoenzyme, comprising RNAP core enzyme
(two a subunits, one b subunit, one b0subunit and one
o subunit) and one of several s factors (16–19). The
primary s factor (e.g. s70in Escherichia coli) is respon-
sible for transcription initiation of genes expressed in ex-
ponentially growing cells, whereas alternative s factors
are responsible for transcription initiation of genes ex-
pressed during stationary phase or in response to specific
*To whom correspondence should be addressed. Tel: +1 978 380 7324; Fax: +1 978 921 1350; Email: email@example.com
Published online 10 May 2010 Nucleic Acids Research, 2010, Vol. 38, No. 13e141
? The Author(s) 2010. Published by Oxford University Press.
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.
conditions (e.g. s32in E. coli for heat-shock response)
(19–21). In vitro reconstitution of E. coli transcription ini-
tiation by conventional methods generally involves expres-
sion 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 sim-
ultaneously 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
The reconstituted protein synthesis system in this study
is derived from PURESYSTEM (4), which comprises
purified E. coli ribosomes and bulk tRNAs, purified re-
combinant E. coli aminoacyl-tRNA synthetases, transla-
tion factors and energy regeneration enzymes, and purified
recombinant T7 RNA polymerase, which couples tran-
scription from a T7 promoter of a DNA template to
protein synthesis system, we co-express Ec RNAP holoen-
zyme 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 add-
promoter (Figure 1). Successful synthesis of a functional
Ec RNAP holoenzyme leads to transcription of the
reporter gene from the sigma-specific promoter and sub-
sequent translation of the reporter protein from the
MATERIALS AND METHODS
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 s70-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
Biotechnology (Madison, WI).
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 (a, b,
b0and o) and E. coli sigma factors (s70and s32) 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 s70and s32, linear DNA tem-
plates were prepared by direct amplification from their
cloning vector pUCA108T7 s70and pUCA108T7 s32, re-
sulting in linear fragments containing the coding regions
for s70and s32with 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 50UTR containing the ribosome-binding site and
30UTR derived from the vector. The promoter region
amplified from E. coli genomic DNA and the promoter
region of tac[...TTGACA(?35)..16nt..TATAAT
(?10)GTGTGGA(+1)] was amplified from pMalp2x
vector (New England Biolabs). The amplification reac-
tions generated a 30sequence that overlapped with the 50
sequence of the reporter DNA fragment, allowing subse-
quent overlapping PCR reactions (second PCR) to yield
the linear DNA templates encoding the reporter gene
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 (a, b, b0and o) and a sigma
factor (s) are encoded in separate linear or plasmid DNA templates
under the control of a T7 promoter (PT7). A reporter, firefly luciferase
(Fluc), is encoded in an additional linear DNA template under the
control of an E. coli promoter (PEc). The synthesized Ec RNAP
subunits are assembled into RNAP core enzyme (Ec RNAP core)
and subsequently form the holoenzyme with the sigma factor (s)
during transcription initiation. rbs: ribosome-binding site; AUG: start
e141Nucleic Acids Research, 2010,Vol.38, No. 13PAGE 2 OF 10
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