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Processivity of proteolytically modified forms of T7 RNA polymerase
D K Muller1, C T Martin, J E Coleman
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Biochemistry
|July 26, 1988
Summary
Proteolytic modification of T7 RNA polymerase affects its DNA binding and processivity. Cleaved forms show altered transcription initiation and RNA binding capabilities, impacting enzyme function.
Area of Science:
- Molecular Biology
- Enzymology
Background:
- T7 RNA polymerase is a crucial enzyme for transcription.
- Proteolytic modifications can alter enzyme activity and function.
Purpose of the Study:
- To characterize two proteolytically modified forms of T7 RNA polymerase.
- To investigate the impact of these modifications on transcription initiation, processivity, and DNA/RNA binding.
Main Methods:
- Characterization of modified T7 RNA polymerase species (80K-20K and 80K).
- Assays for transcription initiation and processivity.
- Gel retention assays for RNA and DNA binding studies.
Main Results:
- The 80K-20K form is active in initiation but shows reduced processivity on long DNA.
- The 80K form initiates transcription similarly to native enzyme but cannot escape abortive cycling.
- Both modified forms bind specifically to promoter DNA, but lack nonspecific DNA binding compared to native enzyme.
- RNA binding is reduced in 80K-20K and absent in 80K species.
Conclusions:
- Proteolytic cleavage significantly impacts T7 RNA polymerase function, particularly processivity and nucleic acid binding.
- The N-terminal 20K domain or a shared region is implicated in nonspecific polynucleotide binding.