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Characterization of a set of T7 RNA polymerase active site mutants
1Department of Biochemistry, University of Texas Health Sciences Center, San Antonio 78284-7819.
Abstract:
We have evaluated the elongation rates, processivities, and abortive transcription characteristics of a set of T7 RNA polymerase mutants that map to the polymerase active site. The effects of these mutations on transcription are complex: they cause decreases in activity and processivity during both the processive and abortive phases of transcription and exhibit disproportionate decreases in activity and processivity on poly(dA).poly(dT) or poly(dT) versus poly(dG).poly(dC) templates. They also exhibit an increase in the proportion of slippage dependent poly(G) transcript synthesis during the initial stages of transcription. It is shown that these multiple, distinct effects on transcription can be attributed to decreases in the mutant enzymes' phosphodiester bond formation rates. Estimates of the decreases in these rates are derived from the measured transcript elongation rates and processivities of the mutant enzymes.
Insights
Mutations in T7 RNA polymerase active site decrease enzyme activity and processivity. These complex effects stem from reduced phosphodiester bond formation rates, impacting RNA synthesis on various DNA templates.
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- T7 RNA polymerase is a key enzyme in transcription.
- Understanding its active site is crucial for enzyme engineering and drug development.
- Mutations can alter enzyme function, but their precise effects are complex.
Purpose of the Study:
- To investigate the impact of mutations in the T7 RNA polymerase active site on transcription.
- To characterize changes in elongation rates, processivity, and abortive transcription.
- To determine the underlying mechanisms responsible for altered enzyme activity.
Main Methods:
- Site-directed mutagenesis of T7 RNA polymerase.
- In vitro transcription assays using various DNA templates (e.g., poly(dA).poly(dT), poly(dG).poly(dC)).
- Measurement of RNA synthesis rates, processivity, and abortive initiation products.
Main Results:
- Mutations led to decreased activity and processivity in both processive and abortive transcription phases.
- Enzyme activity showed disproportionate decreases on specific DNA templates (poly(dA).poly(dT) or poly(dT) vs. poly(dG).poly(dC)).
- Increased slippage-dependent poly(G) transcript synthesis was observed during initial transcription stages.
Conclusions:
- The observed complex transcription defects are attributed to reduced phosphodiester bond formation rates in mutant enzymes.
- Elongation rates and processivity measurements allowed estimation of decreased catalytic rates.
- This study provides insights into structure-function relationships within the T7 RNA polymerase active site.