kathleen big poster 2016 final copy
TRANSCRIPT
Examination of a Potential Open Reading Frame Co-transcribed with DksA in E. coliKathleen 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, weengineered a DNA construct with• a C-terminal His6-tag on ORF • an IPTG-inducible promoter (Ptac) transcribing ORF
Starvation/StressppGpp
Starvation/StressppGpp
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
123450.5TGCAProb
e
Probe
Exp. Stat.
HoursofGrowth
P1
P2
P3
A
B
C
1 k
b la
dd
er
CBA
Acrylamide gel electrophoresis of PCR Products
PCR Strategy
*
*
10
0 b
pla
dd
er
Plasmids carrying Ptac-ORF
Co
ntr
ol P
lasm
id
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 bp500 bp Ptac-ORF-His6
DNA insert
Plasmids carrying Ptac-ORF-His6 DNAverified 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
Size
Sta
nd
ard
s
Lysa
te
Flo
w t
hro
ugh
Was
h
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
Flo
w T
hro
ugh
Pre
cisi
on
Plu
s La
dd
er
Co
ntr
ol L
ysat
e
Pta
c-O
RF-
His
6 L
ysat
e
5
10
15
20
25
37
50
100250
75
MW (kDa)
13 kDa
8.45 kDa expected size
Bound
Size
Sta
nd
ard
s
Co
ntr
ol
Pta
c-O
RF
Pta
c-O
RF
Pta
c-O
RF
Co
ntr
ol
Co
ntr
ol
10 µg 20 µg 30 µg
5
10
15
20
25
37
50
100250
75
MW (kDa)
8.45 kDa expected size
Proteins Expressed in ∆lon Strain Proteins from ∆lon Strain Purifed by
Ni2+ - IMAC Chromatography
FTFT FTFT Was
h
Was
h
Was
h
Was
h
Bo
un
d
Bo
un
d
Bo
un
d
Bo
un
d
Ptac-ORF-His6Control
8.45 kDaexpected 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.