KINETICS OF PROMOTER KINETICS OF PROMOTER ESCAPE VARIES AS A ESCAPE VARIES AS A

FUNCTION OF REACTION FUNCTION OF REACTION CONDITIONSCONDITIONS

Sophiya Karki and Elina Sophiya Karki and Elina ShresthaShrestha

Dr. Lilian Hsu, Biochem Dept.Dr. Lilian Hsu, Biochem Dept.

Summer Science Symposium Summer Science Symposium 20072007

Promoter

N25 N25an

ti

Escape Half life

~3mins

~45mins

Transcription Initiation and Transcription Initiation and Promoter EscapePromoter Escape

The escape rate for N25 >>N25anti

BackgroundBackground

• R+P RPcR+P RPc

• R : RNA Polymerase (RNAP)R : RNA Polymerase (RNAP)• P : Promoter DNAP : Promoter DNA• RPc : RNAP-promoter closed complexRPc : RNAP-promoter closed complex• RPo : Productive RNAP-promoter open complexRPo : Productive RNAP-promoter open complex• RPo' : Unproductive RNAP-promoter open complexRPo' : Unproductive RNAP-promoter open complex• EC : Elongation complexEC : Elongation complex

RPo

RPo’k-2

k2 EC +RNA

kE

KB

Abortive transcripts

Fig 1. Kinetic diagram of Transcription Initiation

Promoters Studied and Past Promoters Studied and Past ObservationObservation

Initial Transcribed Sequences (ITS)Initial Transcribed Sequences (ITS)

N25(-C) Promoter (Escape competent)N25(-C) Promoter (Escape competent)

AUAAAUUUGA GAGAGGAGUU UAAAUAUGGAUAAAUUUGA GAGAGGAGUU UAAAUAUGGCC+1

G29

N25anti(-A) Promoter (Escape incompetent)

GUCCGGCGUC CUCUUCCCGG UCCGUCUGGC UGGUUCUCGC A C40

+1

Promoter

Half life of N25

Amount of full length RNA (fmoles)

Half life of N25anti

Amount of full length

RNA (fmoles)

PL ~3mins ~16 ~45mins ~27

N25anti promoter escapes 10 folds slower than N25 as indicated by the long escape half life but produces higher amounts of full length RNA.

Transcription start site

(Nwe-Nwe Aye-Han, 2007. Senior Thesis)

+3 +20

+20+3

Up stream

ObjectivesObjectives

• To examine if the value of To examine if the value of kk-2-2 is significant for N25 is significant for N25 promoterpromoter

R+P RPcR+P RPc

To study the efficiency of promoter escape for N25 and To study the efficiency of promoter escape for N25 and N25anti promoter under various transcription conditions. N25anti promoter under various transcription conditions.

A= The amount of full length RNA produced

Increasing productive RNA

Hypothetical plot

RPo

RPo’k-2

k2 EC +RNAKB

Abortive transcripts

kE

N25(-C) PromoterN25(-C) Promoter

AUAAAUUUGA GAGAGGAGUU UAAAUAUGGAUAAAUUUGA GAGAGGAGUU UAAAUAUGGCC+1

G29 N25anti(-A) Promoter

GUCCGGCGUC CUCUUCCCGG UCCGUCUGGC UGGUUCUCGC A

C40

+1

Experimental proceduresExperimental procedures

RNAP3’deoxy CTP

Making template for single cycle transcription

• Single cycle transcription (RNAP limiting Single cycle transcription (RNAP limiting condition)condition)

A mix B mix

Transcription Buffer Transcription Buffer

DNA (20nM) A:U:G mix

RNAP [α-32P]UTP

KCl 3’-dCTP

KCl

Incubate A mix at 370C for 10 mins to form open complexes.Transfer the B mix into A mix.At each time points take 5ul aliquots of reaction mixture and add into 5ul FLB to terminate the reaction. 18 time points were taken.

AUAAAUUUGA GAGAGGAGUU UAAAUAUGGC

[α-32P] UTP labeled full length RNA

30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’40’60’90’

Time course Transcription in 200mM KCl of N25 promoterTime Points

dilutions1:301:901:2701:8101:2430

Full length RNA

Abortive RNA

0

5 105

1 106

1.5 106

2 106

2.5 106

3 106

0 20 40 60 80 100

Time course transcription in 200mM KCl

IQV

Time (min)

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

950020m1

436142.4934e+6m2

0.00765320.15663m3

NA1.4441e+11Chisq

NA0.99491R

Postulated plot vs. Experimental plot

A= The amount of full length RNA produced

Increasing productive RNA

Hypothetical plot Experimental plot

Since the plateau level of productive RNA formed remains constant over time, we conclude that the value of k-2 is negligible for N25 promoter.

k-2 ?

Concentration of KCl as a Factor Concentration of KCl as a Factor in Transcription Initiation and in Transcription Initiation and

Promoter EscapePromoter Escape• In steady-state transcription, high salt concentration In steady-state transcription, high salt concentration

(~200mM ) favors the formation of stable open complexes and (~200mM ) favors the formation of stable open complexes and so forth a high yield of full length RNA.so forth a high yield of full length RNA.

• Optimal KCl concentration is different for different promoters.Optimal KCl concentration is different for different promoters.– For N25 it is 250mMFor N25 it is 250mM

– For N25For N25antianti it is 150mM it is 150mM

Current interest :Current interest :

1.1.Variation in escape rate and productive yield as a Variation in escape rate and productive yield as a function of [KCl]. function of [KCl].

2.2.Performed time-course transcription for four [KCL], Performed time-course transcription for four [KCL], 200mM, 100mM, 50mM and 10mM200mM, 100mM, 50mM and 10mM

30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’40’60’90’

Time course Transcription in 200mM KCl of N25 promoterTime Points

dilutions1:301:901:2701:8101:2430

Full length RNA

Abortive RNA

Radioactivity of the reaction mixture from full length RNA is counted by the scintillation counter in cpm (counts per minute) and the RNA bands are measured in Image Quant Volume (IQV) units.

IQV

0

5 105

1 106

1.5 106

2 106

2.5 106

3 106

0 20 40 60 80 100

Time course transcription in 200mM KCl

IQV

Time (min)

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

950020m1

436142.4934e+6m2

0.00765320.15663m3

NA1.4441e+11Chisq

NA0.99491R

Time Course Transcription in 200mM KCl

y = 283.88x - 411936R2 = 0.9999

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

0 50000 100000

150000

200000

250000

300000

cpm

IQV y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

1.1222e+50m1

294211.497e+6m2

0.311691.9222m3

NA2.0149e+11Chisq

NA0.9577R

Dilutions IQV cpmcpm* dilution factor    a value (m3)   

         2493400 277044.44IQV/ trxpt

1:90 70697957 250251 22522590         

1:270 22945676 83246 22476420   slope (IQV/ cpm) 283.88 975.92 cpm/loading

1:810 7752070.7 28032 22705920        

1:2430 2666859.1 9644 23434920    692611.11 IQV in 5 uL rxn mix  

  cpm/10uL 22784963    2439.80 cpm in 5ul rxn mix  

  cpm/uL 2278496.25 

Amount of productive RNA = 21.42 fmoles  

  cpm/fmol 113.92        

Half Life of full length RNA produced in 200mM KCl= 4.0 mins

y= A(1-e-kx)

Time course Transcription in 100mM KCl of N25 promoter 30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’40’60’90’

dilutions1:301:901:2701:8101:2430

Full length RNA

Time pts.

Abortive RNA

30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’ 40’ 60’ 90’

Full length RNA

dilutions1:301:901:2701:8101:2430

Time course Transcription at 50mM KCl of N25 promoter

Time course Transcription at 10mM KCl of N25 promoter 30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’ 40’ 60’ 90’

dilutions1:301:901:2701:8101:2430

Full length RNA

-2 105

0

2 105

4 105

6 105

8 105

1 106

1.2 106

-20 0 20 40 60 80 100

Time couse Transcription in 100mM KCL

IQV

Time (min)

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

936480m1

301211.0177e+6m2

0.100990.73321m3

NA1.4032e+11Chisq

NA0.94762R

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

936480m1

301211.0177e+6m2

0.100990.73321m3

NA1.4032e+11Chisq

NA0.94762R

N25 promoter

Half life=0.9minsAmount of full length RNA producedin 100mM KCl =25.8 fmoles (<60 fmoles )

Half life = 0.5mins

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

4.6524e+50m1

1.3322e+56.7281e+6m2

0.184941.4087m3

NA3.2467e+12Chisq

NA0.96719R

-2 106

0

2 106

4 106

6 106

8 106

-20 0 20 40 60 80 100

Time course Transcription in 50mM KCl

IQV

Time (mins)

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

4.6524e+50m1

1.3322e+56.7281e+6m2

0.184941.4087m3

NA3.2467e+12Chisq

NA0.96719R

Amount of full length RNA producedin 50mM KCl =30.0 fmoles

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

7.1234e+50m1

1.8751e+56.3719e+6m2

0.508441.9988m3

NA7.6114e+12Chisq

NA0.91791R

-2 106

0

2 106

4 106

6 106

8 106

1 107

-20 0 20 40 60 80 100

Time Course Transcription in 10mM KCl

BIQ

V

time (mins)

y = m1 + m2*(1 - exp(-m3*x))

ErrorValue

7.1234e+50m1

1.8751e+56.3719e+6m2

0.508441.9988m3

NA7.6114e+12Chisq

NA0.91791RHalf life = 0.3mins

Amount of full length RNA produced in 10mM KCl= 32.5 fmoles

Future PlansFuture Plans-Try different transcription -Try different transcription conditions other than KCl conditions other than KCl concentration. For example, concentration. For example, changing NTP concentration.changing NTP concentration.

-Further investigation is -Further investigation is required to reach the required to reach the conclusion.conclusion.

 KCl concentration 200mM KCL 100mM KCL 50mM KCL10mM KCl

Half life(mins)

4.42 0.87 0.36 0..34

3.46 0.95 0.5

Amount of full length RNA per loading in fmoles

24.42 24.33 42.6 32.53

 20.50  25.84  30.00

Experimental Results

Lower the concentration of KCl, faster is the escape and higher is the productive yield. This result was completely opposite to what was expected.

AcknowledgementAcknowledgement

Prof. HsuProf. Hsu

Nwe-Nwe Aye-Han, ‘07 graduate Nwe-Nwe Aye-Han, ‘07 graduate

and all other lab membersand all other lab members

Biochem dept.Biochem dept.

Mount Holyoke CollegeMount Holyoke College