Effects of solution conditions on the steady-state kinetics of initiation of transcription by T7 RNA polymerase.
The T7 family of DNA-dependent RNA polymerases presents an ideal model system for the study of fundamental aspects of transcription. The small size of the promoter allows a variety of studies based on simple steady-state kinetics in the synthesis of a five-base runoff transcript. This assay can be used to characterize the effects on the initiation of transcription of simple modifications to potential protein or DNA specificity contacts. In the current work, in vitro conditions for this assay have been identified which optimize the apparent Km for the interaction between the enzyme and the promoter DNA. The addition to the reaction mixture of 0.05% Tween-20 and the substitution of 10 mM NaCl by 100 mM potassium glutamate not only improves the quality of the kinetic assays but also decreases Km by about an order of magnitude (strengthening the interaction between polymerase and its promoter). As observed for DNA binding in other systems, the parameter Km increases substantially with increasing [NaCl], but the salt dependence is shifted to higher concentrations as a function of [KGlu]. Thermal denaturation of the protein, monitored by circular dichroism spectroscopy, confirms the effects of salt and supports a model in which Cl- and other anions compete for phosphate binding sites on the protein. Finally, while Km is highly dependent on [NaCl], the measured kcat is relatively insensitive to salt. These data indicate that the parameters Km and kcat reflect changes respectively in promoter binding and in a rate-limiting step or steps leading to the initiation of transcription.
Maslak
M
M
Department of Chemistry, University of Massachusetts, Amherst 01003-4510.
Martin
C T
CT
D000838:Anions; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D003902:Detergents; D004795:Enzyme Stability; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008274:Magnesium; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012965:Sodium Chloride; D013439:Sulfhydryl Reagents; D014158:Transcription, Genetic / Q000502:physiology*; D014764:Viral Proteins
D000838:Anions; D003902:Detergents; D005971:Glutamates; D013439:Sulfhydryl Reagents; D014764:Viral Proteins; D018698:Glutamic Acid; D012965:Sodium Chloride; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium