1. KINETICS OF PROMOTER ESCAPE VARIES AS A FUNCTION OF KCL CONCENTRATION. Sophiya Karki and Elina Shrestha Dr. Lilian Hsu, Biochem Dept.

2. Background R+P RPc R : RNA Polymerase (RNAP) P : Promoter DNA RPc : RNAP-promoter closed complex RPo : Productive RNAP-promoter open complex RPo' : Unproductive RNAP-promoter open complex EC : Elongation complex RPo RPo’ k -2 k2k2 EC + Full length RNA kEkE KBKB Abortive transcripts Fig 1. Kinetic diagram of Transcription Initiation

3. Promoters Studied and Past Observation Initial Transcribed Sequences (ITS) N25(-C) Promoter (Escape competent) AUAAAUUUGA GAGAGGAGUU UAAAUAUGGC +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 N25 anti 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 +3 Up stream

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5. OBJECTIVE To study the efficiency of promoter escape at various KCl concentrations for the two promoters, N25 and N25anti, differing only in their Initial Transcribed Sequence(ITS).

6. Concentration of KCl as a Factor in Transcription Initiation and Promoter Escape KCl concentration may affect multiple steps during transcription initiation. – Open complex formation, stability and collapse. – Promoter escape Current interest : 1.Observe variation in escape rate and productive yield as a function of [KCl]. 2.Performed time-course transcription for four [KCL], 200mM, 100mM, 50mM and 10mM

7. N25(-C) Promoter AUAAAUUUGA GAGAGGAGUU UAAAUAUGGC +1 G29 N25anti(-A) Promoter GUCCGGCGUC CUCUUCCCGG UCCGUCUGGC UGGUUCUCGC A C40 +1 Experimental procedures RNAP 3’deoxy CTP EXPERIMENTAL SETUP Single-cycle Templates: Template Confirmation: PL: long PCR fragment of 348 bp spanning -234 to +114

8. Single-cycle Transcription reaction The reactions were performed under single-cycle polymerase limiting conditions. The N25 and N25 anti promoters were modified such that they had one of the four nucleotides missing in the first 30 to 40 nucleotides of their initial transcribed sequences. This allows for the halting of polymerase after a round of initiation and elongation when supplied with the 3’-deoxy form of the missing nucleotide, and the rest 3 NTPs. Methodology:

9. In rate diagram, K B is the binding constant that governs the rapid equilibrium of the formation of closed complexes. The figure illustrates the branched pathway model for abortive and productive initiation. The closed ITCs can partition into either productive (RP o ) or unproductive (RP o ’) open complexes. The productive complexes can undergo escape to give rise to full-length RNA (FL), though only after some cycles of abortive initiation. The unproductive complexes are limited to producing abortive transcripts. The synthesis of FL showed a time course of single exponential rise which fit the equation [y=m1 +m2*(1-exp(-m3*x) where m1=0]. The m3 (k E ) and m2 represent the composite rate constant of escape and the plateau level of FL respectively. Extracting these values allowed us to measure the half life of escape (t 1/2 =k E /ln2) and the productive fraction (m2 in fmoles).

10. Single cycle transcription (RNAP limiting condition) A mixB mix Transcription Buffer DNA A:U:G mix(N25)/ G:C:U mix(N25anti) RNAP[α- 32 P]UTP KCl3’-dCTP KCl -Incubate A mix at 37 0 C 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- 32 P] UTP labeled full length RNAInclude in the paragraph

11. 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 promoter Time Points dilutions 1:30 1:90 1:270 1:810 1:2430 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. IQ V

12. Dilutions IQVcpm cpm* dilution factor a value (m3) 2493400277044.44IQV/ trxpt 1:907069795725025122522590 1:270229456768324622476420 slope (IQV/ cpm) 283.88975.92cpm/loading 1:8107752070.72803222705920 1:24302666859.1964423434920 692611.11 IQV in 5 uL rxn mix cpm/10uL22784963 2439.80 cpm in 5ul rxn mix cpm/uL2278496.25 Amount of productive RNA =21.42 fmoles cpm/fmol113.92 Half Life of full length RNA produced in 200mM KCl= 4.0 mins y= A(1-e -kx )

13. Experimental Results In N25 promoters, lower the concentration of KCl, faster is the escape and higher is the productive yield. In N25anti promoters, lower the concentration of KCl, higher is the productive yield. However, the rate of escape is NOT increasing as in N25. [KCl]Productive Yield (fmoles)Half-life(mins) 200mM20.96 (0.65)3.95 (0.68) 100mM25.67 (1.26)0.80 (0.19) 50mM36.15 (9.21)0.43 (0.09) 10mM32.530.35 [KCl]Productive Yield (fmoles)Half-life(mins) 200mM4.96 (6.97)24.33 (7.12) 150mM11.43 (5.98)48.95(9.03) 100mM21.71 (8.18)47.44 (8.41) 50mM13.91 (8.03)96.58 (16.19) 10mM19.10 (11.93)69.22 (15.31) N25 promoterN25 anti promoter

14. Graphical Overview

15. Conclusion KCl concentration influences the partitioning step in transcription initiation similarly in N25 and N25 anti promoters. KCl concentration affects the rate of promoter escape in N25 and N25 anti promoters differently since rate of escape is highly governed by their ITSs.

16. N25 / [KCl] Amount of RNAHalf-life R value Gel No. 200mM21.424.430.965 20.503.470.988 20.963.95 σ= 0.65 σ= 0.68 100mM24.330.870.926 25.840.95 7 26.830.580.8910 25.670.80 σ= 1.26 σ= 0.19 50mM42.660.360.969 29.630.490.9712 36.150.43 σ= 9.21 σ= 0.09 10mM32.530.350.9211 Observed data for N25

17. N25anti Amount of RNA(fmoles) Half- life(mins) R value Gel No. 200 mM(Nwe- Nwe) 27.2 (8.5)41.9 (24.0) 200mM0.9813.350.9716 1.6721.640.9018 3.0526.360.9720 17.3531.41 0.9830 1.7428.900.98 32 4.9624.33 σ=6.97σ=7.12 150mM8.7551.440.9922 7.2656.470.9629 18.2838.940.9931 11.4348.95 σ=5.98σ=9.03 100mM13.0638.860.9917 22.7647.790.9919 29.3255.670.9921 21.7147.44 σ=8.18σ=8.41 50mM20.2581.070.9923 16.6195.300.9926 4.88113.380.9928 13.9196.58 σ=8.03σ=16.19 10mM27.5358.390.9825 10.6680.050.9927 19.1069.22 σ=11.93σ=15.31

18. U14 C10 G8 C7 G6 G5 C4 U12 N25anti (A-):PL t=3-270 min 50mM KCl A41

19. U14 C10 G8 C7 G6 G5 C4 C3 U2 U12U12 U12U12 U12 N25anti (A-): PL A41 t=3-270 min 10mM KCl t=3-270 min A41

20. U14 C10 G8 C7 G6 G5 C4 U12 N25anti (A-) :PL t=3-270min 100mM KCl A41

21. U14 C10 G8 C7 G6 G5 C4 C3 U2 U12 N25anti (A-):PL t=3-270min 150mM KCl A41

22. U14 C10 G8 C7 G6 G5 C4 C3 U2 U12 N25anti(A-):PL t=3-270min 200mM KCl A41

23. G11 G9 U8 U7 U6 A5 C4 C3 U2 C30 N25 (-C):PL T=3-90min 200mM KCl