1. Examination of a Potential Open Reading Frame Co-transcribed with DksA in E. coli
Kathleen Barakat and Robert Osuna
Department of Biological Sciences, University at Albany, 1400 Washington Avenue, Albany, NY 12222
ABSTRACT
DksA is a critical transcription factor in Escherichia coli that plays an essential role in the response to
conditions of stress. DksA is an uncommon transcriptional regulator in that it binds directly to RNA
polymerase (RNAP) and not to DNA. Recent research has focused on acquiring detailed information on
how E. coli cells control the production of DksA in different growth conditions. There are three major
temporal promoters transcribing the dksA gene: P1, is highly expressed during early to mid exponential
growth phase, P2 is transiently expressed during entry into stationary phase, and P3 is a stationary phase-
specific promoter. The P3 promoter is dependent on the RpoS stress-dependent sigma factor. It is located
within the sfsA gene and is followed by what might possibly be a small open reading frame (ORF) of
unknown function. The goal of my work is to determine whether or not this ORF produces a protein
product that myight be co-expressed with dksA during stationary phase. To accomplish this, the segment
of DNA that transcribes the ORF was amplified by the polymerase chain reaction (PCR) such that a His6-
tag was added onto the C-terminal end of the putative ORF. The PCR product was purified, ligated on a
multicopy plasmid, and transformed into a wild-type E. coli strain. We then attempted to induce the
expression of the His6-tagged protein and purify it using Ni+-based affinity chromatography. Our results
repeatedly failed to detect induced levels of ORF polypeptide in different strains of E. coli. We therefore
reject the hypothesis that the putative open reading frame is translated into a polypeptide in vivo. These
results help clarify our picture of the roles played by the dksA promoters.
BACKGROUND
DksA is a transcription regulator that acts together with ppGpp
to mediate the Stringent Response
DksA Mediates its Effects By Binding to RNA Polymerase
CONCLUSION
RESULTS
Several temporal promoters transcribe dksA.
Hypothesis: The putative ORF encodes a polypeptide, which is co-expressed
with DksA during stationary phase
Matches to known Helix-Turn-Helix DNA binding motifs
MSVAAEGQRAVIFFAVLHSAITRFSPARHIDEKYAQLLSEAQQRGVEILAYKAEISAEGMALKKSLPVTL
I A S V A Q H V C L S P S R L S H L F R AraC
L Y D V A E Y A G V S Y Q T V S R V V N LacR
Q T K T A K D L G V Y Q S A I N K A I H Cro
T R K L A Q K L G V E Q P T L Y W H V K TetR
I K D V A R L A G V S V A T V S R V I N GalR
R A E I A Q R L G F R S P N A A E E H L LEXA
L L S E A Q Q R G V E I L A Y K A E I S ORF
Predicted ORF Polypeptide of 70 Amino Acids
Significance: Co-transcribed genes in bacteria are often involved in related
functions. As a putative polypeptide with a DNA-binding motif, the product of
ORF may be envisioned to collaborate with DksA in regulating gene expression
during stationary phase.
Approach:
To investigate whether ORF can be translated in vivo into a polypeptide, we
engineered a DNA construct with
• a C-terminal His6-tag on ORF
• an IPTG-inducible promoter (Ptac) transcribing ORF
Starvation/Stress
ppGpp
Starvation/Stress
ppGpp
Secondary Channel
Secondary Channel
DksA
DksA
Transcription Activation
Transcription Repression
DksA
ppGpp
ppGpp
Primer Extension Analysis
P2
P3
P1
S1-Nuclease Analysis
-154
-53
-475
1 2 3 4 50.5TGCA
Probe
Probe
Exp. Stat.
Hours of Growth
P1
P2
P3
A
B
C
1kbladder
CBA
Acrylamide gel electrophoresis
of PCR Products
PCR Strategy
*
*
100bpladder
Plasmids carrying Ptac-ORF
ControlPlasmid
5% Polyacrylamide Gel of candidate plasmids
carrying Ptac-ORF. Digestion with EcoRI and
HindIII releases a 340 bp DNA fragment containing
the Ptac-ORF-His6 construct.
Ni2+-IMAC Chromatography of Native Proteins
100 bp
200 bp
300 bp
400 bp
500 bp Ptac-ORF-His6
DNA insert
Plasmids carrying Ptac-ORF-His6 DNA
verified by restriction digestion
sfsA
sfsA
Grow Cells carrying:
-Plasmid with Ptac-ORF-His6
-Control Plasmid
Induce with IPTG
for 2 hours at 37°C
Collect and Lyse Cells
Bind to Ni2+-IMAC Affinity
Chromatography Column
Flow
Through
Wash Bound
Experimental Outline
Bound
SizeStandards
Lysate
Flowthrough
Wash
MW (kDa)
75
150
5
10
15
20
25
37
50
100
250
13 kDa
8.45 kDa expected size
Ni2+-IMAC Chromatography of Denatured Proteins
FlowThrough
PrecisionPlusLadder
ControlLysate
Ptac-ORF-His6Lysate
5
10
15
20
25
37
50
100
250
75
MW (kDa)
13 kDa
8.45 kDa expected size
Bound
SizeStandards
Control
Ptac-ORF
Ptac-ORF
Ptac-ORF
Control
Control
10 µg 20 µg 30 µg
5
10
15
20
25
37
50
100
250
75
MW (kDa)
8.45 kDa expected size
Proteins Expressed in ∆lon Strain
Proteins from ∆lon Strain Purifed by
Ni2+ - IMAC Chromatography
FT
FT
FT
FT
Wash
Wash
Wash
Wash
Bound
Bound
Bound
Bound
Ptac-ORF-His6Control
8.45 kDa
expected size
We see no evidence of Expression of a polypeptide of the expected size for OR(8.5 kDa)
after IPTG induction from a strong promoter and after several attempts to capture the
polypeptide using Ni2+ - IMAC affinity chromatography. Therefore, we reject the
hypothesis that ORF expresses a polypeptide in vivo. These results help clarify our picture
of the possible roles of the dksA promoters.