kinetics of promoter escape varies as a function of reaction conditions
Post on 31-Jan-2016
46 Views
Preview:
DESCRIPTION
TRANSCRIPT
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
top related