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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: P 1 , is highly expressed during early to mid exponential growth phase, P 2 is transiently expressed during entry into stationary phase, and P 3 is a stationary phase- specific promoter. The P 3 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 MSVAAEGQRAVIFFAVLHSAITRFSPARHIDEKYAQ LLSEAQQRGVEILAYKAEIS AEGMALKKSLPVTL 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 P 2 P 3 P 1 S1-Nuclease Analysis -154 -53 -475 12345 0.5 T G C A Probe Probe Exp. Stat. Hours of Growth P 1 P 2 P 3 A B C 1 kb ladder C B A Acrylamide gel electrophoresis of PCR Products PCR Strategy * * 100 bp ladder Plasmids carrying Ptac-ORF Control Plasmid 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. Ni 2+ -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 Size Standards Lysate Flow through Wash MW (kDa) 75 150 5 10 15 20 25 37 50 100 250 13 kDa 8.45 kDa expected size Ni 2+ -IMAC Chromatography of Denatured Proteins Flow Through Precision Plus Ladder Control Lysate Ptac-ORF-His6 Lysate 5 10 15 20 25 37 50 100 250 75 MW (kDa) 13 kDa 8.45 kDa expected size Bound Size Standards 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 Ni 2+ - IMAC Chromatography FT FT FT FT Wash Wash Wash Wash Bound Bound Bound Bound Ptac-ORF-His6 Control 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 Ni 2+ - 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.

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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.