Nucleic Acids Research, 1990, Vol. 18, No. 12 3445-3450
© 1990
ENZYMOLOGY |
A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase
Department of Biochemistry, College of Medicine, East Tennessee State University Johnson City, TN 37614, USA
*To whom correspondence should be addressed
Received May 4, 1990. Revised May 18, 1990. Accepted May 18, 1990.
A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min. on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44°C. The thermostability at 44°C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about 4 fold at 37°C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed.
+Present address: Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616, USA