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The functional role of basic patch, a structural element of Escherichia coli transcript cleavage factors GreA and
1Department of Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, New York 11203, USA.
The Journal of Biological Chemistry
|April 25, 2000
Summary
The GreA and GreB proteins regulate transcription elongation in E. coli. Their N-terminal basic patch is crucial for anchoring RNA, enabling readthrough and anti-arrest functions essential for in vivo activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- GreA and GreB are essential transcript cleavage factors in Escherichia coli, regulating transcription elongation.
- Structural analysis revealed distinct N-terminal basic patches on GreA and GreB.
Purpose of the Study:
- To investigate the functional role of the N-terminal basic patch in GreA and GreB proteins.
- To determine how modifications to the basic patch affect transcript cleavage and transcription regulation.
Main Methods:
- Engineering and biochemical characterization of mutant Gre proteins with altered basic patch size and charge.
- Assessing binding to RNA polymerase, transcript cleavage efficiency, and photocross-linking to RNA.
- Complementation assays in a thermosensitive E. coli strain.
Main Results:
- Mutants lacking or with reduced basic patches showed similar binding and initial cleavage activity but reduced readthrough and cleavage of arrested complexes.
- Basic patch mutants exhibited decreased photocross-linking efficiency to the RNA 3'-terminus.
- Basic patch-less mutants failed to complement the thermosensitive phenotype of GreA(-):GreB(-) E. coli.
Conclusions:
- The basic patch is not directly involved in inducing transcript cleavage but is essential for anchoring nascent RNA.
- This RNA anchoring is critical for the readthrough, anti-arrest, and in vivo functions of Gre factors.