title: 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.
authors: Biophysics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India.
authors: Sudipta
authors: S
authors: Pal
authors: Dipak
authors: D
authors: Dasgupta
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D061785:Transcription Initiation, Genetic; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases