Functional map of the alpha subunit of Escherichia coli RNA polymerase. Deletion analysis of the amino-terminal assembly domain.
The alpha subunit of Escherichia coli RNA polymerase plays a major role in the assembly of the core enzyme. The amino-terminal two-thirds of the alpha subunit, as far as position 235, are involved in this assembly. To define the site(s) within this region required for core enzyme assembly, we constructed a set of amino-terminal and internal deletion mutants of the rpoA gene. The overexpressed alpha derivatives were purified to apparent homogeneity and examined for their abilities to assemble beta and beta' subunits into active core enzymes in vitro. Among a total of 22 alpha derivatives tested, only four mutants retained the activity form active core enzyme. These mutants had deletions of the extreme amino-terminal residues as far as amino acid residue 30. The minimum fragment with full activity of the core assembly was alpha(21 to 235), with deletions of 20 amino-terminal and 94 carboxy-terminal amino acid residues. Most of the other mutants appeared to be defective in the formation of stable alpha dimers as analyzed by high-pressure liquid chromatography gel filtration, although some formed self-aggregates. These results, taken together, suggest that the amino-terminal region of the alpha subunit with the core assembly activity is highly structured, and any deletion within this domain disrupts its ordered conformation. Deletions of the extreme amino-terminal region did not affect transcription activation by CRP at the lacP1 promoter or by OmpR at the ompC promoter.
Kimura
M
M
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
Fujita
N
N
Ishihama
A
A
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D012321:DNA-Directed RNA Polymerases