Differential scanning calorimetric approach to study the effect of melting region upon transcription initiation by T7 RNA polymerase and role of high affinity GTP binding

Abstract

Transcription initiation by T7 RNA polymerase is a multistep process consisting of the transition from closed to open complex. The promoters of bacteriophage T7 share a consensus sequence of 23 base pairs, from −17 to +6, relative to transcription start site (+1). In the present study, we have characterized T7 RNA polymerase-promoter complexes by means of fluorescence spectroscopy and differential scanning calorimetry. We have examined the effect of high affinity GTP binding upon the equilibrium of the transition from closed to open complex. We have employed the promoter containing 23 base pair consensus sequence and two variants containing Adenine-Thymine and Guanine-Cytosine stretches in the melting region of the promoter sequence. Variation in the nucleotide sequence of melting region does not have any effect upon the affinity of promoter-T7 RNAP complex. On the other hand, alteration of the base sequence in the melting region of the promoter affects the isomerization process among the closed and open complexes. When the initiating nucleotide GTP is prebound to T7 RNA Polymerase, the isomerization process is affected only in case of the promoter with consensus sequence.

Keywords:

• promoter melting
• thermal stability
• differential scanning calorimetry

Acknowledgements

The authors acknowledge the financial support from an intramural grant, CBAUNP, from Department of Atomic Energy, India. We acknowledge Parijat Majumder for her suggestions and critical reading of the manuscript.