The effect of a nested set of C-terminal substituted deletions on the function of the alpha subunit of Escherichia coli RNA polymerase.
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 codon +1 orf extending from within codon L262 and terminating 16 bp downstream of the rpoA reading frame. These frameshifts give rise to a set of substituted alpha deletions that are all of the same size (334 aa) and carry segments of the Orf sequence replacing the alpha 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 alpha 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 alpha subunit does not prevent formation of a transcriptionally proficient holoenzyme form of RNA polymerase capable of complementing rpoA112(Ts); (ii) the extreme C-terminal Orf region, like that of alpha itself, is exposed in holoenzyme; (iii) these substituted deletions are not commonly functional at class I activated promoters.
Thomas
M S
MS
Division of Genetics, School of Clinical Laboratory Sciences, Queen's Medical Centre, Nottingham University, U.K.
Zou
C
C
Ishihama
A
A
Glass
R E
RE
D000483:Alleles; D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D017384:Sequence Deletion
D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases