Functional map of the alpha subunit of Escherichia coli RNA polymerase: insertion analysis of the amino-terminal assembly domain.
The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. Deletion analysis of alpha indicated that the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 is involved in this assembly. For fine mapping of the site(s) within this region required for subunit-subunit contacts, we constructed a set of insertion mutants of the rpoA gene, each encoding mutant alpha with two extra amino acid residues, Ala (A) and Ser (S), inserted at 20 residue intervals. The over-expressed alpha derivatives were purified to apparent homogeneity and examined for their ability to form dimers and to assemble beta and beta' subunits into core enzymes in vitro. Among a total of 11 alpha insertion derivatives tested, four mutants having the insertion at dispersed positions retained the ability to form active core enzymes. Other mutants showed defects in core enzyme assembly at various steps depending on the position of AS insertion: one mutant formed an unstable alpha 2 beta complex; one mutant exhibited decreased binding of beta' subunit; and five mutants did not form stable alpha dimers, of which one formed an alpha 2 beta complex and another formed an alpha beta complex. These results suggest that alpha dimerization involves multiple contact sites. Among alpha mutants with dimer formation ability, the mutation at amino acid residue 80 interfered with the binding of both beta and beta' subunits, and the mutation at position 200 made the alpha dimer inactive in beta' binding.
Kimura
M
M
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
Ishihama
A
A
D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial / Q000235:genetics; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D012333:RNA, Messenger; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases