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Pausing and termination by bacteriophage T7 RNA polymerase
1Morse Institute for Molecular Genetics, Health Science Center at Brooklyn, State University of New York, 450 Clarkson Avenue, Brooklyn, Box 44, NY 11203-2098, USA.
Journal of Molecular Biology
|July 9, 1998
Summary
Bacteriophage T7 RNA polymerase (RNAP) pausing and termination at class II sites are influenced by the ATCTGTT sequence and U-runs. T7 lysozyme may inhibit RNAP reversion to elongation, impacting termination.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Bacteriophage T7 RNA polymerase (RNAP) exhibits pausing and termination at specific DNA sites.
- Two classes of termination sites (Class I and Class II) have been identified, with distinct mechanisms.
- Class II sites, unlike Class I, do not form stable RNA stem-loops but possess conserved sequences and U-runs.
Purpose of the Study:
- To analyze pausing and termination at Class II sites and their variants.
- To elucidate the role of the conserved sequence and U-runs in Class II termination.
- To investigate the influence of T7 lysozyme on RNAP pausing and termination.
Main Methods:
- Analysis of pausing and termination at various Class II sites and their mutants.
- Investigated the effect of the 7 bp sequence ATCTGTT on RNAP activity.
- Studied the impact of U-runs and T7 lysozyme on termination efficiency.
Main Results:
- The 7 bp sequence ATCTGTT (5' to 3') acts as a pausing signal for T7 and T3 RNA polymerase.
- Termination efficiency at Class II sites is enhanced by U-runs, potentially by destabilizing RNA:DNA hybrids.
- T7 lysozyme's effects suggest termination involves RNAP reversion to initiation, with lysozyme inhibiting return to elongation.
Conclusions:
- The ATCTGTT sequence is crucial for pausing at Class II termination sites.
- U-runs facilitate termination by destabilizing the RNA:DNA hybrid.
- A kinetic model explains pausing and termination, incorporating RNAP conformational changes and T7 lysozyme activity.