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In vitro characterization of transcription termination factor Rho from Escherichia coli rho(nusD) mutants
1Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140 USA.
Journal of Molecular Biology
|July 19, 1996
Summary
Mutations in the transcription termination factor Rho (nusD strains) in Escherichia coli impair phage growth. Mutant Rho proteins exhibit altered RNA binding and termination activity, impacting bacteriophage T4 development.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli nusD strains possess mutations in the rho gene, affecting transcription termination factor Rho.
- These mutations are known to inhibit the growth of bacteriophages T4 and lambda r32.
Purpose of the Study:
- To identify and characterize rho mutations in nusD strains.
- To investigate the functional impact of these mutations on Rho protein activity and bacteriophage growth.
Main Methods:
- Genetic analysis to identify rho mutations in six independent nusD strains.
- Overexpression, purification, and biochemical characterization of five mutant Rho proteins.
- In vitro transcription assays using lambda tR1 and cro terminators.
Main Results:
- Five distinct mutations were identified in the proposed RNA-binding domain of Rho.
- Mutant Rho proteins retained RNA-dependent ATPase activity but showed reduced termination efficiency at the lambda tR1 terminator.
- A novel Rho-dependent terminator (tRE) was identified within the cro gene, with termination sites distinct from tR1.
Conclusions:
- Mutations in the Rho RNA-binding domain alter its termination function.
- The identified rho mutations likely interfere with bacteriophage T4 growth via indirect effects on host gene expression.