The effect of a nested set of C-terminal substituted deletions on the function of the α subunit of Escherichia coli RNA polymerase

Abstract A genetic screen was devised to obtain plasmid-borne rpoA alleles exhibiting partial or no complementation of the chromosomal Escherichia coli rpoA341 allele responsible for a pleiotropic phenotype. Nine of the ten mutants obtained carried single base pair deletions within the 3′ end of rpoA resulting in frameshifting into a 72 colon + 1 orf extending from within colon L262 and terminating 16 by downstream of the rpoA reading frame. These frameshifts give rise to a set of substituted a deletions that are all of the same size (334 aa) and carry segments of the Orf sequence replacing the a region from the C-terminus (residue 329) to various points between 272 > 319. The in vivo properties of this nested set of nine C-terminal-substituted derivatives of the a subunit of RNA polymerase have been assessed in terms of their assembly and transcriptional proficiency. The results indicate: (i) replacement of as much as 42 C-terminal residues of the a subunit does not prevent formation of a transcriptionally proficient holoenzyme form of RNA polymerase capable of complementing rpoA 112(Ts); (ii) the extreme C-terminal Orf region, like that of α itself, is exposed in holoenzyme; (iii) these substituted deletions are not commonly functional at class I activated promoters.