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Kinetic and thermodynamic studies of initiation of transcription by T7 RNA polymerase
Abstract
T7 RNA polymerase is a single subunit DNA-dependent RNA polymerase which is highly specific for the initiation of transcription from a relatively small consensus promoter sequence. The simplicity of this system makes it an ideal model system in which to better understand the enzymology of transcription, including the chemical basis of promoter recognition and the initiation of transcription. Interactions between T7 RNA polymerase and its promoter DNA have been studied by a steady-state kinetic assay and by thermodynamic methods. The in vitro reaction conditions for initiation have been optimized to include a nonionic detergent Tween-20 and the replacement of NaCl by potassium glutamate. The specific incorporation of deoxyuridine in place of thymidine at individual sites in the promoter results in the replacement of an exocyclic methyl group by hydrogen and so provides a probe of potential contacts. Many of these substitutions do not affect binding or catalysis, however, the thymine methyl at position $-6$ is critical to recognition. Two methyl groups near the start site on the template strand also contribute to promoter specificity, while nearby methyl groups on the nontemplate strand do not, suggesting interactions with only the template strand near the start site. To test this hypothesis, kinetic parameters associated with a variety of templates in which the nontemplate strand is partially deleted near the start site have been compared. The results suggest that in this region, promoter recognition involves a form of the promoter DNA which is melted near the start site for transcription and the enzyme interacts with only the template strand. The temperature dependence of the initiation kinetics shows a single apparent activation energy of 26 kcal/mol for the fully duplex promoter, and studies with partially single stranded constructs show that less than 10% of this barrier is related to melting of the downstream promoter region. These results have led to a revised model for promoter recognition, characterized by specific binding to a form of the promoter which is duplex upstream of about position $-6$ and melted downstream through the start site. Within the melted region, the polymerase interacts significantly only with the template strand of the promoter DNA.
Subject Area
Chemistry|Biochemistry|Biophysics
Recommended Citation
Maslak, Maribeth, "Kinetic and thermodynamic studies of initiation of transcription by T7 RNA polymerase" (1994). Doctoral Dissertations Available from Proquest. AAI9420659.
https://scholarworks.umass.edu/dissertations/AAI9420659