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Kinetics of Promoter escape varies as a function of reaction Conditions

Kinetics of Promoter escape varies as a function of reaction Conditions. Sophiya Karki and Elina Shrestha Dr. Lilian Hsu, Biochem Dept. Summer Science Symposium 2007. Transcription Initiation and Promoter Escape. Background. k E. K B. RPo. EC +RNA. k 2. R+P RPc

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Kinetics of Promoter escape varies as a function of reaction Conditions

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  1. Kinetics of Promoter escape varies as a function of reaction Conditions SophiyaKarki and ElinaShrestha Dr. Lilian Hsu, Biochem Dept. Summer Science Symposium 2007

  2. Transcription Initiation and Promoter Escape

  3. Background kE KB RPo EC +RNA k2 • 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 k-2 RPo’ Abortive transcripts Fig 1. Kinetic diagram of Transcription Initiation

  4. Promoters Studied +1 G29 N25anti(-A) Promoter GUCCGGCGUC CUCUUCCCGG UCCGUCUGGC UGGUUCUCGC A +1 C40 Initial Transcribed Sequences (ITS) N25(-C) Promoter AUAAAUUUGA GAGAGGAGUU UAAAUAUGGC

  5. Observations from the Past Experiments N25anti (-A) N25 (-C) A= amount of full length RNA formed (Nwe-New Aye-Han, 2007)

  6. Objective RPo RPo’ k2 RPc k-2 • To examine if the value of k-2is significant for N25 promoter • To study the efficiency of promoter escape for N25 and N25anti promoter under various transcription conditions.

  7. k-2 is negligible for N25 The amount of full length RNA produced remained constant

  8. Concentration of KCl as a Factor in Transcription Initiation and Promoter Escape • In steady-state transcription, high salt concentration (~200mM ) favors the formation of stable open complexes and so forth a high yield of full length RNA. • In lower concentration of KCl, we expected to see lower yield.

  9. Time course Transcription in 200mM KCl of N25 promoter Time Points 30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’40’60’90’ dilutions 1:30 1:90 1:270 1:810 1:2430 Full length RNA Abortive RNA

  10. Half Life of full length RNA produced in 200mM KCl= 4.24 mins y= A(1-e-kx)

  11. Time course Transcription in 100mM KCl of N25 promoter Time pts. 30’’ 1’ 1.5’ 2’ 2.5’ 3’ 3.5’ 4’ 4.5’ 5’ 7’ 10’ 15’ 20’ 30’40’60’90’ dilutions 1:30 1:90 1:270 1:810 1:2430 Full length RNA Abortive RNA

  12. N25 promoter Half life=0.95mins Amount of full length RNA produced in 50mM KCl =25.8 fmoles

  13. Time course Transcription at 50mM 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’ dilutions 1:30 1:90 1:270 1:810 1:2430 Full length RNA

  14. Half life = 0.5mins Amount of full length RNA produced in 50mM KCl =30.0 fmoles

  15. 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’ dilutions 1:30 1:90 1:270 1:810 1:2430 Full length RNA

  16. Half life = 0.34mins Amount of full length RNA produced in 10mM KCl= 32.5 fmoles

  17. Experimental Results

  18. Future Plans -Try different transcription conditions other than KCl concentration. For example, changing NTP concentration. -Further investigation is required to reach the conclusion.

  19. Acknowledgement Prof. Hsu Nwe-Nwe Aye-Han, ‘07 graduate and all other lab members Biochem dept. Mount Holyoke College

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