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Applied and Environmental Microbiology, December 2000, p. 5399-5405, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Coordinated, Differential Expression of Two Genes
through Directed mRNA Cleavage and Stabilization by Secondary
Structures
Christina D.
Smolke,
Trent A.
Carrier,
and
J. D.
Keasling*
Department of Chemical Engineering,
University of California, Berkeley, California 94720-1462
Received 14 July 2000/Accepted 29 September 2000
 |
ABSTRACT |
Metabolic engineering and multisubunit protein production
necessitate the expression of multiple genes at coordinated levels. In
bacteria, genes for multisubunit proteins or metabolic pathways are
often expressed in operons under the control of a single promoter; expression of the genes is coordinated by varying transcript stability and the rate of translation initiation. We have developed a system to
place multiple genes under the control of a single promoter and produce
proteins encoded in that novel operon in different ratios over a range
of inducer concentrations. RNase E sites identified in the
Rhodobacter capsulatus puf operon and Escherichia
coli pap operon were separately placed between the coding regions
of two reporter genes, and novel secondary structures were engineered into the 5' and 3' ends of the coding regions. The introduced RNase E
site directed cleavage between the coding regions to produce two
secondary transcripts, each containing a single coding region. The
secondary transcripts were protected from exonuclease cleavage by
engineered 3' secondary structures, and one of the secondary transcripts was protected from RNase E cleavage by secondary structures at the 5' end. The relative expression levels of two reporter genes
could be varied up to fourfold, depending on inducer concentration, by
controlling RNase cleavage of the primary and secondary transcripts. Coupled with the ability to vary translation initiation by changing the
ribosome binding site, this technology should allow one to create new
operons and coordinate, yet separately control, the expression levels
of genes expressed in that operon.
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INTRODUCTION |
In prokaryotes, expression of genes
for multistep pathways or for production of multisubunit proteins is
often controlled by regulating the posttranscriptional processing of a
polycistronic mRNA containing the coding regions for all the enzymes in
that pathway or all subunits in a multisubunit protein. This type of control eliminates the need for multiple promoters of different strengths for each gene. To produce enzymes at appropriate levels, the
cell balances translation efficiency with the rate that the message is
inactivated by RNases.
These nucleases can be classified into two categories by comparing
their mechanisms of action and role in mRNA decay. Two endonucleolytic
activities are responsible for bulk mRNA processing
RNase III and
RNase E (3, 10). RNase III cleaves mRNA at a weak consensus
sequence within double-stranded regions of an RNA (9, 31).
This indicates the role of RNase III in RNA processing, as it can
cleave stem regions of RNA secondary structures (30, 31).
RNase E is responsible for bulk inactivation of mRNA (14). The nuclease scans the mRNA transcript in a 5'-to-3' direction and
cleaves within AU-rich segments (1, 23, 24, 34). It is
generally recognized that RNase E requires a free 5' end to bind to the
mRNA before scanning the transcript for cleavage sites (11-13,
15). The two exonucleases responsible for bulk mRNA degradation
into mononucleotides are RNase II and polynucleotide phosphorylase
(25). These enzymes degrade mRNA in a processive 3'-to-5'
direction, which indicates their role in degradation rather than
inactivation (1).
For the majority of mRNA species, the initial cleavage within a
transcript functionally inactivates the mRNA and is followed by
3'-to-5' exonuclease activity (2). It has been shown that hairpin structures at the 3' end protect the mRNA from degradation by
exoribonucleases (1, 10, 26), and hairpins at the 5' end
protect from initial inactivation by endoribonucleases
(11-13). Although there are a number of commercial products
that contain 3' hairpins to protect mRNA from exonucleases, there have
been few attempts to design 5' hairpins and recognition sites for
endoribonucleases to alter mRNA stability and affect gene expression
(2, 6, 7, 12, 13).
An expression system was developed that allows for the introduction of
synthetic DNA cassettes into the region between the transcription and
translation start sites of a gene of interest. Upon transcription, the
5' end of the mRNA corresponding to the synthetic cassette forms a
hairpin that protects the mRNA from endonucleolytic cleavage (6,
7). Depending on the structure of the inserted hairpin, the
hairpin-containing mRNA exhibits half-lives between 3 and 10 times that
of the mRNA with no hairpin, resulting in increases in mRNA and protein
levels (8). These results indicate that it is possible to
engineer mRNA stability as an additional means of controlling gene expression.
In this work, we constructed an expression system that allows one to
vary the expression levels of two genes, both of which are under the
control of the same promoter, by introducing DNA cassettes encoding
mRNA secondary structures and RNase cleavage sites at various locations
in the operon. The design for this decoupled, dual-gene expression
system was derived from two native systems: the puf
operon of Rhodobacter capsulatus (1, 21, 22) and the pap operon of Escherichia coli
(5, 29). The expression system was tested with a combination
of mRNA secondary structures at the 5' and 3' ends of the two coding
regions and putative RNase E cleavage sites between the coding regions
to determine if it is possible to independently vary expression of the
two genes. Northern blotting and primer extension analysis indicate
that the primary transcript was cleaved into two secondary transcripts,
each containing a coding region, when a putative RNase E site was
placed between the coding regions. Northern blotting and enzyme assays
indicate that expression of the two genes was effectively decoupled and
that the relative expression levels of the two genes varied with the
mRNA secondary structures and RNase E sites in the operon.
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MATERIALS AND METHODS |
Bacterial strains, media, chemicals, and enzymes.
E.
coli DH10B (Gibco BRL) was used for all cloning steps. E. coli JS4 (Bio-Rad) was used for Northern blot analysis and enzyme assays (Table 1).
Luria-Bertani (LB) medium was made as described by Maniatis et al.
(
32). C medium (
18) was supplemented with 3.4%
glycerol,
1.0% Casamino Acids, and micronutrients (
28).
Ampicillin, used
at a concentration of 100 µg/ml, and arabinose were
purchased
from Fisher Scientific. Diethyl pyrocarbonate and rifampin
were
obtained from
Sigma.
Restriction enzymes were purchased from Roche and New England Biolabs.
T4 DNA ligase and High-Fidelity PCR enzyme mix were
obtained from
Roche.
Pfu Turbo polymerase, DNase, ribosomal RNasin,
SP6
RNA polymerase, and avian myeloblastosis virus reverse transcriptase
were purchased from Promega. T4 polynucleotide kinase was obtained
from
New England Biolabs. DNA sequencing kits were purchased from
USB.
Plasmid construction.
The base dual-gene plasmid containing
both gfp and lacZ (p50gl) was constructed in
several cloning steps. Primers used in each PCR step were synthesized
by Genemed Synthesis, Inc. DNA amplification was performed with
High-Fidelity PCR enzyme mix. All PCRs were performed with 20 mM
Tris-HCl (pH 8.3), 30 mM KCl, 1.5 mM MgCl2, 200 µM
deoxynucleoside triphosphates (dNTPs), 300 µM primers, 0.5 µg of
template, and 2.6 U of enzyme. Cycle times and temperatures followed
those suggested by the enzyme manufacturer. All plasmids used in this
study are described in Table 1.
PCR was performed on pGFPuv (Clonetech) using primer 1 (5'-TGCCTGCAGGTCGACTCTAGAGGATCCCC-3') and primer 2 (5'-TCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTC-3')
to remove
the
SalI restriction site in
gfpuv and to replace
codons
to improve translation efficiency. The 669-bp PCR product and
plasmid pGFPuv were digested with
PstI and
BstBI
and ligated to
form pGFPuvm by using T4 DNA ligase. The ligation
products were
electroporated into
E. coli DH10B. The
transformation products
were plated onto LB agar plates containing
ampicillin, arabinose,
and
5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside (X-Gal)
(Gibco
BRL). To determine if the
SalI site was present in
pGFPuvm, plasmid
was isolated from various colonies (
32),
digested with
SalI,
and analyzed by agarose gel
electrophoresis.
A second round of PCR was performed on pGFPuvm using primers 1 and 10 (5'-GATGTATACATTGTGTGAGTTATAGTTGTACTCCAGTTTGTGTCCGAGAA-3')
to remove the
XhoI restriction site in
gfpuvm and to replace codons
to improve translation
efficiency. The 496-bp PCR product was
substituted for the
XhoI-containing fragment in pGFPuvm to form
pGFPuvm2 by
digesting both vector and insert with
PstI and
Bst1107I
and ligating the two DNA fragments. Screening for
pGFPuvm2 was
performed by digesting with
XhoI.
PCR was also used to replace the
Asp718 restriction site in
pTC40 with
NheI using primer 11 (5'-CTCCATACGTCGACAGCTAGCGTATTTTGGATG-3')
and primer 12 (5'-GTCGGTTTATGCAGCAACGAGACGTCAC-3'). The 675-bp
PCR product
was cloned into pTC40 to form pTC40m by digesting
both vector and
insert with
NheI and
AatII and ligating the two
fragments. Screening for pTC40m was performed by digesting with
NheI.
The final step in the construction of the dual-gene operon was to place
lacZ after
gfp using splicing by overlap
extension
(
19,
20). DNA amplification was performed with
Pfu Turbo polymerase.
Primer 13 (5'-TGGATGATAACGAGGCGCAAAAAATGAGTAAAGGAGAAGAACTTTTCACT-3')
and primer 4 (5'-AGCGGTACCAGCAGATCTTATTTGTAGAGCTCATCCATGCC-3')
were designed to amplify
gfp from template
pGFPuvm2, while primers
14 (5'-CGCTAACCAAACCGGTAACC-3') and
15 (5'-TTTTTGCGCCTCGTTATCATCCA-3')
were designed to amplify
lacZ from template pTC40m. During the
splicing by overlap
extension, the two amplified segments connected
to form a single 999-bp
product. This product was cloned into
pTC40 to form p50gl by digesting
both vector and insert with
BstEII
and
Asp718 and
ligating the two fragments. Cells that fluoresced
were screened for
p50gl.
The
gfp single-gene control plasmid (p50g
l) was
constructed by digesting p50gl with
PvuII and purifying by
agarose gel electrophoresis
and self-ligating the 5,559-bp product. The
correct plasmid was
found using blue/white screening by plating
transformation products
on LB agar plates containing arabinose and
X-Gal and selecting
white
colonies.
The plasmid used to produce the
gfp probe (pCS01) was
constructed by digesting pGFPuvm2 and pGEM-4z (Promega) with
EcoRI and
Asp718. The 550-bp insert was ligated
into pGEM-4z. The correct
plasmid was found using blue/white
screening.
The control plasmid for the primer extension studies (pCS02) was
constructed by digesting p60gHP41 and pGEM-4z with
PstI and
SalI. The 4,000-bp insert containing
gfp and
lacZ was ligated
into pGEM-4z. The correct plasmid was found
using blue/white
screening.
DNA cassettes.
The various DNA cassettes were synthesized
(Genemed Synthesis, Inc.) as two complementary DNA oligonucleotides.
These oligonucleotides were annealed at high concentration by heating
to above their annealing temperature and ramping the temperature down
to 20°C. The DNA cassettes were inserted into the plasmid using
unique restriction sites at the ends of the cassettes and within the plasmid itself. The plasmid and the cassettes were digested and ligated
together. The ligation products were electroporated into E. coli DH10B. The transformed cells containing the plasmid with the
cassette insert were screened using a restriction site within the cassette.
Enzyme assays.
To determine -galactosidase and green
fluorescent protein (GFP) activities, C medium was inoculated with a
stock culture to an optical density at 600 nm (OD600) of
0.0015 and grown at 30°C. At an OD600 of 0.20, samples
were removed and placed on ice. -Galactosidase assays were performed
as described previously (6, 27). -Galactosidase activity
is reported in Miller units. GFP activity was determined by measuring
the relative fluorescence of a 2-ml sample in a VersaFluor fluorometer
(Bio-Rad). This fluorescent reading was divided by the
OD600 reading for the sample to obtain the GFP specific activity.
Northern blot analysis.
Northern blot analysis was performed
to determine transcript stabilities. C medium was inoculated with a
stock culture of E. coli JS4 containing the various plasmids
to an OD600 of 0.016 and grown at 30°C. At an
OD600 of 0.05, arabinose was added to a final concentration
of 0.1%. At an OD600 of 0.20, rifampin was added to a
final concentration of 2 mg/ml. RNA extraction was performed on samples
as described previously at an OD600 of 0.20 (6).
The
lacZ and
gfp probes were synthesized by
digesting pTC01 and pCS01 with
PvuII and
NcoI.
The fragments were separately combined
with SP6 polymerase and
radiolabeled [
-
32P]CTP (Amersham) and nonradiolabeled
ribonucleotide triphosphates
(Promega).
Prior to the Northern blotting, all equipment was treated with 3%
hydrogen peroxide and all solutions were treated with dimethyl
pyrocarbonate. Northern blot analysis was conducted as described
previously (
2). Labeled probe was added to the hybridization
solution at a concentration of 10
6 cpm/ml. Membranes were
hybridized with 5 ml of the probe hybridization
solution. The blots
were visualized using a PhosphorImager (Molecular
Dynamics). The
intensities were recorded using IPLab Gel software
and were plotted as
a function of time for several different exposure
times to ensure
linearity of the band intensity with exposure
time. Northern blots were
repeated at least twice on samples to
ensure reproducibility.
Uncertainties in half-life calculations
were determined according to
the method described by Taylor (
33).
Half-life calculations.
Half-lives of the various
transcripts were determined according to the following model:
![<FR><NU>d[P]</NU><DE>dt</DE></FR>=k<SUB>1</SUB>[X]−k<SUB>dp</SUB>[P]](/content/vol66/issue12/fulltext/5399/img001.gif)
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(1)
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![<FR><NU>d[S<SUB>1</SUB>]</NU><DE>dt</DE></FR>=k<SUB>dp</SUB>[P]−k<SUB>ds<SUB>1</SUB></SUB>[S<SUB>1</SUB>]](/content/vol66/issue12/fulltext/5399/img002.gif)
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(2)
|
![<FR><NU>d[S<SUB>2</SUB>]</NU><DE>dt</DE></FR>=k<SUB>dp</SUB>[P]−k<SUB>ds<SUB>2</SUB></SUB>[S<SUB>2</SUB>]](/content/vol66/issue12/fulltext/5399/img003.gif)
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(3)
|
where [
P] is the concentration of the primary
transcript, [
S1] and
[
S2] are the concentrations of the two
secondary transcripts,
kdp is the decay constant
for the primary transcript,
kds1 and
kds2 are the decay constants of the secondary
transcripts, and
k1[
X] is the rate of
synthesis of the primary transcript. This
model assumes that the
processing of the primary transcript is
the only source of the
secondary transcripts and that decay of
the transcripts is proportional
to the concentrations of the transcripts
present. In the experiments
reported here,
k1[
X] is set equal
to zero,
because transcription is stopped with rifampin. Thus,
the half-life of
the primary transcript can be calculated from
the simplified form of
equation
1.
We determined the half-lives of the secondary transcripts using two
different methods and assumptions. The first method assumes
that
kdp is much larger than
kds1 and
kds2 or that the
primary
transcript is degraded fast relative to the secondary
transcripts.
With this assumption, the source term for the secondary
transcripts
in equations 2 and 3 can be ignored relative to the decay
term.
The half-lives of the secondary transcripts can be calculated
from the simplified form of equations 2 and
3.
The assumptions made in the first method are correct only when no
message is being produced (that is, for stable secondary
transcripts
and relatively unstable primary transcripts). The
second method used to
determine half-lives of the secondary transcripts
makes no assumptions
about the stability of the primary transcript
and solves the equations
using forward differences. This method
provides more accurate half-life
values for secondary transcripts
arising from primary transcripts that
have approximately the same
half-life.
Primer extension analysis.
An oligonucleotide, RT-primer 1 (5'-CGACGGGATCTGCGATAGCTGTC-3'), was synthesized (Genemed
Synthesis, Inc.) to bind 50 nucleotides downstream of the suspected
RNase E cleavage site location. RT-primer 1 was labeled at its 5' end
with [
-32P]ATP (ICN Biochemicals) using T4
polynucleotide kinase. The labeled oligonucleotide, at a concentration
of 106 cpm/µl, was annealed to 25 µg of the RNA
isolated from the cellular extract (6). RNA samples were
taken at an OD600 of 0.20. Primer extension was performed
using avian myeloblastosis virus reverse transcriptase and 5 µg of
total RNA in 50 mM KCl, 12 mM DTT, 3 mM spermidine, 10 mM
MgCl2, and 1.1 mM dNTPs. The reactions were stopped after
30 min with an 8 M urea-0.1% sodium dodecyl sulfate stop
buffer-denaturing buffer solution. To determine the locations of the
stop bands, a DNA sequencing ladder was generated by annealing the
labeled oligonucleotide at a concentration of 5 × 105
cpm/µl to 5 µg of the corresponding denatured plasmid. Plasmid DNA
was denatured in a 0.2 M NaOH-0.2 mM EDTA solution at 37°C for 30 min. Denatured DNA was neutralized by adding sodium acetate to a final
concentration of 0.3 M. The dideoxynucleotide (Sanger) method of
sequencing was performed using Sequenase Kit Version 2.0. Protocol for
sequencing followed that provided by the manufacturer. These samples
were separated in individual lanes on a denaturing 7 M urea-6%
polyacrylamide gel in Tris-borate-EDTA (Stratagene CastAway Sequencing
System). This 16- by 7-in. gel was run for 1.5 h at 40 W and then
dried for 30 min.
The sequence was read from the gel by exposing it to film for an
appropriate amount of time and developing the film. The film
was
scanned into Adobe Photoshop using VistaScan software. Because
the
amount of RNA that could be added to the reactions was limited
by
poisoning of the primer extension reaction from the total RNA
present
in the samples, it was not possible to vary the intensity
of the primer
extension bands. Therefore, bands for primer extension
and DNA
sequencing differed greatly in intensity, and the images
were adjusted
separately. Image intensities were adjusted, first
to read the sequence
ladders. The intensities of the bands in
the lanes containing the three
reverse transcriptase reactions
were adjusted equally to visualize the
bands in these lanes. These
increased intensity lanes were placed next
to their corresponding
DNA sequencing ladder and lower intensity lanes
on the original
image for
comparison.
Cleavage locations were determined where reverse transcriptase would no
longer extend the transcript. To ensure that the stop
bands were not a
result of secondary structure, the procedure
was repeated with 10 ng of
RNA synthesized in vitro. In vitro-synthesized
RNA was obtained by
digesting pCS02 with
PvuII. The fragments
were combined with
SP6 RNA polymerase and ribonucleotide
triphosphates.
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RESULTS AND DISCUSSION |
Design of dual- and single-gene systems.
A synthetic operon
containing two genes was designed to allow introduction of secondary
structures and RNase cleavage sites in the form of DNA cassettes at
various locations in the operon and easy evaluation of the effects of
these changes on gene expression. The system was constructed in a
high-copy-number plasmid that has an arabinose-inducible
araBAD promoter (PBAD) and ampicillin resistance
marker (Fig. 1A). The gfp gene
was placed upstream of the lacZ gene. Unique restriction
sites were placed at the 5' end of each untranslated region for the
introduction of DNA cassettes that, when transcribed, would form
secondary structures. These sites were placed out of range of the
ribosome binding sites so that secondary structures would not interfere
with translation efficiency. To ensure that there would be no
differences in translation initiation, the regions between the unique
restriction sites and the translation start sites in the two genes were
identical. The restriction sites at the 5' end of gfp allow
one to introduce the hairpin at the very beginning of the transcript,
as it has been reported that more than five unpaired bases at the 5'
end of a transcript will relieve the stability enhancements of the hairpins (4). Hairpins at the 3' end of lacZ
protect the mRNA against degradation by 3'-to-5' exonucleases and
terminate transcription.
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FIG. 1.
Plasmid and DNA cassette design. (A) Backbone structure
of plasmid. All plasmids in this study have the same backbone and
differ only in the variable region. Some examples of RNA
representations of the variable region for various plasmids are shown
in panel C. (B) RNase E sites obtained from literature. E1 was taken
from the puf operon of R. capsulatus and was used
in the p60 constructs, and E2 was taken from the pap operon
of E. coli and was used in the p70 constructs. (C) Schematic
for inserting control elements. RNA representations of the variable
regions for several plasmids are shown, including steps taken to insert
control elements. p50gl is the basic dual-gene plasmid to which
stabilizing elements were added. p50gl lacks the lacZ
coding region. p60gl and p70gl differ in the RNase E site that was
added 5' of lacZ. p50HP4gl, p50HP17gl, p60gHP4l, and
p70gHP4l are the previously mentioned constructs with hairpins inserted
5' of the various genes.
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The decay characteristics of
lacZ are known from previous
work (
6,
7). An understanding of the decay characteristics
for
gfp alone was required. By removing the
lacZ
coding region
from the construct described above, an expression system
with
the
gfp gene alone was developed. This single-gene
system had
5' restriction sites for the insertion of DNA cassettes and
the
same transcription terminators positioned at the 3' end of
gfp.
Design of DNA cassettes.
Two DNA cassettes encoding previously
designed hairpin structures (HP17 and HP4) (8) were inserted
into the single-gene gfp system (Fig. 1). Three DNA
cassettes were inserted sequentially into the dual-gene system. A DNA
cassette encoding a 3' hairpin for gfp and containing an
intercistronic region with a putative RNase E site immediately 5' of
lacZ was inserted into the dual-gene plasmid p50gl, making
plasmid p60gl. This cassette was based on the sequence between the
pufQ and pufB genes of R. capsulatus, which has been shown to contain an RNase E site (16). The
second DNA cassette encoded a 5' hairpin for lacZ and was
inserted immediately downstream of the putative RNase E site, making
plasmid p60gHP4l. The design of this hairpin was based upon HP4
(8). The third DNA cassette encoded a known RNase E site
found between the papA and papB regions of the
E. coli pap operon (29) and was inserted to
replace the first intercistronic region, making plasmid p70gHP4l (Fig.
1).
Transcript stability analysis.
Northern blot analysis
performed on the single-gene gfp systems (p50gl,
p50HP4gl, and p50HP17gl) revealed that the half-life of the
gfp transcript was unaffected by 5' hairpins (Table
2). This result indicates that
gfp is not susceptible to inactivation by an
endoribonuclease, such as RNase E. In contrast, similar hairpins placed
at the 5' end of lacZ significantly affected its stability
(6).
Northern blot analysis conducted on the dual-gene systems revealed
useful qualitative and quantitative information. No stable
intermediate
transcripts were detected for p50gl when probed with
either
gfp or
lacZ (Fig.
2B and
3B).
The primary transcript containing
the
lacZ and
gfp coding regions (Fig.
2B and
3B, band a) was slightly
larger (by the length of
gfp) than the
lacZ-only
transcript of
pHP4l (Fig.
2A, band b). As there was no 5' hairpin to
protect
the primary transcript, it was rapidly degraded. As there were
no internal hairpins (5' to
lacZ), no stable secondary
transcripts
resulted or can be seen. The primary transcript had a short
half-life
of approximately 2 min, since the 5' end of this transcript
was
not protected by secondary structures (Table
2).
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FIG. 2.
Northern blot analysis of lacZ mRNA
stability. Each lane has 5 µg of total RNA loaded for each sample
taken at a particular time after addition of rifampin. Lane 1, 0 min;
lane 2, 2.5 min; lane 3, 5 min; lane 4, 10 min; lane 5, 20 min.
Markers: a, primary transcript; b, secondary lacZ
transcript; c, 23S rRNA; d, 16S rRNA. (A) Blot of pHP4l (single-gene
lacZ system) probed with lacZ. (B) Blot of p50gl
probed with lacZ. There is a gradual smearing under the
primary band, indicating that there is no stable secondary
lacZ transcript. (C) Blot of p60gHP4l with lacZ.
In contrast to panels A and B, there are a dark primary band and a
secondary band running under the primary transcript (corresponding to a
stable secondary lacZ transcript).
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FIG. 3.
Northern blot analysis of gfp mRNA stability.
Each lane has 5 µg of total RNA loaded for a sample taken at a
particular time after addition of rifampin: lane 1, 0 min; lane 2, 2.5 min; lane 3, 5 min; lane 4, 10 min; lane 5, 20 min. Markers: a, primary
transcript; b, secondary gfp transcript; c, 23S rRNA; d, 16S
rRNA. (A) Blot of p50HP4gl (single-gene gfp system)
probed with gfp. (B) Blot of p50gl probed with
gfp. There is a dark smearing where the gfp
secondary transcript should run, indicating that there is no stable
secondary gfp transcript. (C) Blot of p60gHP4l probed with
gfp. In contrast to panels A and B, there are a dark primary
band (a) and a secondary band (b) corresponding to a stable secondary
gfp transcript.
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The results of the Northern blot analysis for p50gl can be compared to
those obtained with p60gHP4l. The blots for this system
revealed bands
for both the primary and secondary transcripts
for
lacZ and
gfp (Fig.
2C and
3C), indicating that after the initial
RNase cleavage between the genes, the resulting transcripts were
protected at vulnerable ends from RNases. The
lacZ secondary
transcript
of p60gHP4l (Fig.
2C, band b) was darker and more distinct
than
that of p50gl (Fig.
2B, band b). Given the size of the bands,
cleavage must occur approximately upstream of the
lacZ 5'
hairpin.
The 3' hairpin for
gfp protects against exonuclease
activity.
(Note that the secondary
gfp transcript from
p60gHP4l is smaller
than that from p50g
l because it lacks the 5' end
of
lacZ and
the 3' hairpins on p50g
l.) The half-life of
the primary transcript
was approximately 3.5 min. The half-lives of the
lacZ and
gfp secondary transcripts were
approximately 3.5 and 3 min, respectively,
from method 1 and 2.9 min
for both from method 2 (Table
2). Note
that the half-lives for the
gfp secondary transcript from p60gHP4l
and p70gHP4l were
significantly shorter than those for the transcripts
of p50g
l,
p50HP4g
l, and p50HP17g
l. The secondary
gfp transcripts
of p60gHP4l and p70gHP4l carry smaller hairpins 3' of
gfp,
in
contrast to p50g
l, p50HP4g
l, and p50HP17g
l, which carry the
large hairpins. The differences in the hairpins at the 3' end
could
significantly affect 3'-to-5' processing and thus
stability.
Northern blot analysis for p70gHP4l showed qualitative trends similar
to those of p60gHP4l. The half-lives of the primary
transcript and the
gfp secondary transcript were similar (within
experimental
error) to those for p60gHP4l (Table
2). In contrast,
the half-life of
the
lacZ secondary transcript was approximately
7 min
according to method 1 for determining half-lives and 5.4
min according
to method 2, nearly double that for p60gHP4l. The
higher stability of
the
lacZ secondary transcript in p70gHP4l
may be the result
of the putative RNase E site in p70gHP4l being
more susceptible to
cleavage by
E. coli RNase E than the one in
p60gHP4l
(
17).
The stability analysis for these systems is complicated by the fact
that the
lacZ secondary transcript is close in size to
the
primary transcript and runs directly beneath the primary band
on the
gel, making the two bands appear as one longer band on
the Northern
blot. The break between the two bands can be determined
by comparing it
to the single-gene blots (Fig.
2A and
3A).
Primer extension analysis.
To confirm that the mRNAs of
p60gHP4l and p70gHP4l were cleaved between the two coding regions and
that two secondary transcripts were present, the nuclease cleavage site
location between the two genes in p60gHP4l and p70gHP4l was determined
using primer extension analysis (Fig. 4).
For p60gHP4l, cleavage occurred approximately 10 bases from the base of
the hairpin stem 5' of the lacZ coding region in the mRNA
(indicated by band 2 in Fig. 4A). For p70gHP4l, cleavage occurred
approximately 5 bases from the base of the hairpin stem 5' of the
lacZ coding region in the mRNA (band 2 in Fig. 4A). For
p50gl, no band appeared on the gel, indicating that cleavage does not
occur between the two coding regions. This analysis indicates that the
system is working as designed and suggests that a stable secondary
transcript containing lacZ is formed when the primary transcript of p60gHP4l and p70gHP4l is cleaved by an RNase. Presumably this RNase is RNase E, as the regions were designed with previously identified RNase E sites. However, the actual identity of the RNase
responsible for the processing is not critical. What is critical is
that cleavage occurs between the two coding regions, thereby
stabilizing the coding regions for the downstream gene. The higher
stability of the lacZ secondary transcript from p70gHP4l than that for the transcript from p60gHP4l may be due to the number of
the 5' unpaired bases. The p60gHP4l lacZ secondary
transcript contained approximately 10 unpaired bases, whereas the
p70gHP4l transcript had approximately 5 unpaired bases. It has been
shown that having more than 5 unpaired bases at the 5' end of a
transcript alleviates the protective effect of the secondary structure
(4). Cleavage closer to the hairpin 5' of lacZ
results in a more stable lacZ transcript and more
-galactosidase produced from p70gHP4l than from p60gHP4l.
View larger version (50K):
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|
FIG. 4.
Primer extension analysis of mRNA from p50gl, p60gHP4l,
and p70gHP4l. (A) Sequencing ladders and primer extension lanes are
shown for each plasmid. The first four lanes of each set are the
plasmid sequencing results. G, ddGTP included in the reaction; A,
ddATP; T, ddTTP; C, ddCTP. The two lanes to the right of the sequencing
ladder are the primer extension results for the in vivo mRNA. N, no
dideoxynucleotides included in the reaction; *, lane N with intensity
magnified to visualize bands. Bands 1 and 2 are reverse transcriptase
stop sites. (B) Sequence of p50gl in this region (no secondary
structures or cleavage sites). (C) Location of the cleavage site
relative to the predicted hairpin structure in p60gHP4l. (D) Location
of the cleavage site relative to the predicted hairpin structure in
p70gHP4l. Note that the sequence read from the gel is the complement to
that shown in panels B to D.
|
|
Note that the larger bands (indicated by the numeral 1 in Fig.
4A) in
the primer extension lanes for p60gHP4l and p70gHP4l
are due to reverse
transcriptase stopping at the secondary structure
3' of the
gfp coding region; p50gl lacks this secondary structure
and
this band. It should also be pointed out that the intensity
of this
band, resulting from the secondary structure, was higher
in both
systems than that of the band corresponding to the cleavage
site. These
relative intensities indicate relative abundance of
mRNA transcripts;
more full-length transcript than processed transcript
was present in
the
sample.
Protein production.
Enzyme assays were conducted on the
extracts of cells carrying the dual-gene operon to determine whether
the changes in transcript stability resulted in changes in protein
production. Expression was induced over a range of inducer
concentrations (Fig. 5A). Cells harboring
p60gHP4l produced twice as much -galactosidase as cells harboring
p50gl across all inducer concentrations. Cells harboring p70gHP4l had
approximately 50 times more -galactosidase at low inducer
concentrations and 2 times more -galactosidase at high inducer
concentrations than did p50gl.
View larger version (22K):
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|
FIG. 5.
Enzyme activities at various inducer concentrations. (A
and B) Triangles, p70gHP4l; squares, p60gHP4l; diamonds, p50gl. (A)
-Galactosidase activity. (B) GFP activity. (C) Relative ratios.
Black bars, -galactosidase/GFP enzyme activity ratio for each
construct relative to p50gl; gray bars, ratio of lacZ to
gfp secondary transcript half-lives for p60gHP4l and
p70gHP4l calculated using method 1; white bars, ratio of
lacZ to gfp secondary transcript half-lives for
p60gHP4l and p70gHP4l calculated using method 2. Since p50gl has no
secondary transcripts, half-life ratios could not be provided.
|
|
Assays of GFP showed a trend similar to that found in the
-galactosidase assays (Fig.
5B). At inducer concentrations below
0.001%, cells harboring p70gHP4l produced approximately 10-fold
more
GFP than cells harboring p60gHP4l and 20-fold more than cells
harboring
p50gl. At inducer concentrations above 0.001%, cells
harboring
p70gHP4l produced about the same amount of GFP as cells
harboring
p60gHP4l, which was approximately twofold greater than
cells harboring
p50gl.
To judge how the secondary structures and putative RNase E cleavage
sites affected mRNA stability and protein production,
the ratios of
half-lives and enzyme activities for one construct
relative to another
are presented. At concentrations of inducer
of less than 0.001% (in
the linear range of inducer concentrations),
the ratios of
-galactosidase-to-GFP activities for p70gHP4l and
p60gHP4l relative
to the same for p50gl,
(
-gal/GFP)
p70/(
-gal/GFP)
p50,
and
(
-gal/GFP)
p60/(
-gal/GFP)
p50 were
consistently 3.5 and 1.7,
respectively (Fig.
5C). The ratios of the
lacZ secondary transcript
half-life to
gfp
secondary transcript half-life (based on values
obtained using method 1 of the half-life calculations) for these
systems were 1.8 and 1.2, respectively. (The secondary transcripts
of p50gl were nonexistent.)
This can be compared to the ratios
for the values obtained using method
2 of the half-life calculations,
1.7 and 1.0, respectively. As the only
difference between these
two constructs was the putative RNase E site,
this result may
reflect the relative efficiency with which RNase E
cleaved the
RNase E site (
16,
29). Indeed, the results of
the enzyme assay
indicate that p70gHP4l contains an RNase site upstream
of the
lacZ gene that is more susceptible to cleavage by an
RNase than
that in p60gHP4l. The secondary
lacZ transcripts,
and to some
extent the
gfp transcripts, from p60gHP4l and
p70gHP4l are more
stable than the primary transcript of p50gl. Cleavage
by an RNase
directly 5' of the 5' hairpin of
lacZ results in
a secondary transcript
with hairpins at the 5' and 3' ends to protect
against further
immediate RNase cleavage. While larger than the
lacZ secondary
transcript by the addition of
gfp
at the 5' end, the primary transcript
of p50gl is not protected from
RNase attack by any 5' hairpins.
This stabilization results in more
-galactosidase production
relative to GFP production from p60gHP4l
and p70gHP4l than from
p50gl. These results show that this technology
can be used to
differentially control the stabilities of secondary
transcripts,
resulting in differential gene expression. More
importantly, the
relative ratios of the enzymes produced can be
affected with this
type of
control.
| |
ACKNOWLEDGMENTS |
We thank N. Pace and S. Kustu for their help with the primer
extension work and C. Bertozzi and P. Schultz for use of their PhosphorImager.
This research was supported in part by the ERC Program of the National
Science Foundation under award number EEC-9731725, National Science
Foundation grants BES-9502495 and BES-9906405, and a National Science
Foundation graduate fellowship to C. D. Smolke.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemical Engineering, University of California, 201 Gilman Hall,
Berkeley, CA 94720-1462. Phone: (510) 642-4862. Fax: (510) 643-1228. E-mail: keasling{at}socrates.berkeley.edu.
Present address: Bacterial Vaccine Technology, Merck and Co.,
Inc., West Point, Pa.
| |
REFERENCES |
| 1.
|
Alifano, P.,
C. Bruni, and M. Carlomagno.
1994.
Control of mRNA processing and decay in prokaryotes.
Genetica
94:157-172[CrossRef][Medline].
|
| 2.
|
Arnold, T. E.,
J. Yu, and J. Belasco.
1998.
mRNA stabilization by the ompA 5' untranslated region: two protective elements hinder distinct pathways for mRNA degradation.
RNA
4:319-330[Abstract].
|
| 3.
|
Belasco, J., and G. Brawerman.
1993.
Control of messenger RNA stability.
Academic Press, New York, N.Y.
|
| 4.
|
Bouvet, P., and J. Belasco.
1992.
Control of RNase E-mediate RNA degradation by 5'-terminal base pairing of E. coli.
Nature
360:488-491[CrossRef][Medline].
|
| 5.
|
Bricker, A., and J. Belasco.
1999.
Importance of a 5' stem-loop for longevity of papA mRNA in Escherichia coli.
J. Bacteriol.
181:3587-3590[Abstract/Free Full Text].
|
| 6.
|
Carrier, T. A., and J. D. Keasling.
1997.
Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5' cassette system.
Biotechnol. Bioeng.
55:577-580[CrossRef].
|
| 7.
|
Carrier, T. A., and J. D. Keasling.
1997.
Controlling messenger RNA stability in bacteria: strategies for engineering gene expression.
Biotechnol. Prog.
13:699-708[CrossRef][Medline].
|
| 8.
|
Carrier, T. A., and J. D. Keasling.
1999.
Library of synthetic 5' secondary structures to manipulate mRNA stability in Escherichia coli.
Biotechnol. Prog.
15:58-64[CrossRef][Medline].
|
| 9.
|
Chelladuri, B.,
H. Li, and A. Nicholson.
1991.
A conserved sequence element for ribonuclease III processing signals is not required for accurate in vitro enzymatic cleavage.
Nucleic Acids Res.
19:1759-1766[Abstract/Free Full Text].
|
| 10.
|
Duetscher, M., and J. Zhang.
1990.
Ribonucleases, diversity and regulation, p. 1-11.
In
J. McCarthy, and M. Tuite (ed.), Post-transcriptional control of gene expression. Springer-Verlag, Berlin, Germany.
|
| 11.
|
Ehrettsmann, C.,
A. Carpousis, and H. Krisch.
1992.
mRNA degradation in prokaryotes.
FASEB J.
6:3186-3192[Abstract].
|
| 12.
|
Emory, S., and J. Belasco.
1990.
The ompA 5' untranslated RNA segment functions in E. coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency.
J. Bacteriol.
172:4472-4481[Abstract/Free Full Text].
|
| 13.
|
Emory, S.,
P. Bouvet, and J. Belasco.
1992.
A 5' terminal stem-loop structure can stabilize mRNA in E. coli.
Genes Dev.
6:135-148[Abstract/Free Full Text].
|
| 14.
|
Fritsch, J.,
R. Rothfuchs,
R. Rauhut, and G. Klug.
1995.
Identification of an mRNA element promoting rate-limiting cleavage of the polycistronic puf mRNA in Rhodobacter capsulatus by an enzyme similar to RNase E.
Mol. Microbiol.
15:1017-1029[Medline].
|
| 15.
|
Hansen, M.,
L. Chen,
M. Fejzo, and J. Belasco.
1994.
The ompA 5' untranslated region impedes a major pathway for mRNA degradation in E. coli.
Mol. Microbiol.
12:707-716[CrossRef][Medline].
|
| 16.
|
Heck, C.,
R. Rothfuchs,
A. Jager,
R. Rauhut, and G. Klug.
1996.
Effect of the pufQ-pufB intercistronic region on puf mRNA stability in Rhodobacter capsulatus.
Mol. Microbiol.
20:1165-1178[CrossRef][Medline].
|
| 17.
|
Heck, C.,
E. Evguenieva-Hackenberg,
A. Balzer, and G. Klug.
1999.
RNase E enzymes from Rhodobacter capsulatus and Escherichia coli differ in context- and sequence-dependent in vivo cleavage within the polycistronic puf mRNA.
J. Bacteriol.
181:7621-7625[Abstract/Free Full Text].
|
| 18.
|
Helmstetter, C., and S. Cooper.
1968.
DNA synthesis during the division cycle of rapidly growing Escherichia coli.
J. Mol. Biol.
31:507-510[CrossRef][Medline].
|
| 19.
|
Horton, R. M.,
Z. Cai,
S. N. Ho, and L. R. Pease.
1990.
Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.
BioTechniques
8:528-535[Medline].
|
| 20.
|
Horton, R. M.
1993.
In vitro recombination and mutagenesis of DNA.
Methods Mol. Biol.
15:251-261.
|
| 21.
|
Klug, G.,
S. Jock, and R. Rothfuchs.
1992.
The rate of decay in Rhodobacter capsulatus-specific puf mRNA segments is differentially affected by RNase E activity in E. coli.
Gene
121:95-102[CrossRef][Medline].
|
| 22.
|
Klug, G.
1993.
The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes.
Mol. Microbiol.
9:1-7[CrossRef][Medline].
|
| 23.
|
Lundberg, U.,
A. von Gabain, and O. Melefors.
1990.
Cleavages in the 5' region of the ompA and bla mRNA control stability: studies with an E. coli mutant altering mRNA stability and a novel endoribonuclease.
EMBO J.
9:2731-2741[Medline].
|
| 24.
|
McDowall, K.,
S. Lin-Chao, and S. Cohen.
1994.
A+U content rather than a particular nucleotide order determines the specificity of RNase E cleavage.
J. Biol. Chem.
269:10790-10796[Abstract/Free Full Text].
|
| 25.
|
McLauren, R.,
S. Newbury,
G. Dance,
H. Causton, and C. Higgins.
1991.
mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo.
J. Mol. Biol.
221:81-95[Medline].
|
| 26.
|
Mejia, J.,
M. Burnett,
H. An,
W. Barnell,
K. Keshav,
T. Conway, and L. Ingram.
1992.
Coordination of expression of Zymomona mobilis glycolytic and fermentative enzymes: a simple hypothesis based on mRNA stability.
J. Bacteriol.
174:6438-6443[Abstract/Free Full Text].
|
| 27.
|
Miller, J.
1992.
A short course in bacterial genetics.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 28.
|
Neidhardt, F.,
P. Bloch, and P. Smith.
1974.
Culture medium for enterobacteria.
J. Bacteriol.
119:736-747[Abstract/Free Full Text].
|
| 29.
|
Nilsson, P., and B. E. Uhlin.
1991.
Differential decay of a polycistronic Escherichia coli transcript is initiated by RNase E-dependent endonucleolytic processing.
Mol. Microbiol.
5:1791-1799[Medline].
|
| 30.
|
Portier, C.,
L. Dondon,
M. Manago, and P. Regnier.
1987.
The first step in the functional inactivation of the E. coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5' end.
EMBO J.
6:2165-2176[Medline].
|
| 31.
|
Regnier, P., and M. Grunberg-Manago.
1990.
RNase III cleavages in non-coding leaders of E. coli transcripts control mRNA stability and genetic expression.
Biochimie
72:825-834[Medline].
|
| 32.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 33.
|
Taylor, J. R.
1982.
An introduction to error analysis, 2nd ed.
University Science Books, Sausalito, Calif.
|
| 34.
|
Zubiaga, A.,
J. Belasco, and M. Greenberg.
1995.
The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.
Mol. Cell. Biol.
15:2219-2230[Abstract].
|
Applied and Environmental Microbiology, December 2000, p. 5399-5405, Vol. 66, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
