A nested set of C-terminal deletions of the alpha subunit of Escherichia coli RNA polymerase define regions concerned with assembly, proteolysis, stabilization and transcriptional activation in vivo.
BACKGROUND
The alpha subunit of eubacterial RNA polymerase comprises an N-terminal assembly domain and a mobile C-terminal domain which provides an activation contact site for class I transcription activators. One particular C-terminal alpha mutant, rpoA341, impairs the response of Escherichia coli RNA polymerase to several activators, including MelR.


RESULTS
The in vivo properties of a set of C-terminally truncated alpha variants were investigated. Derivatives of 230 amino acids or longer were assembled into functional RNA polymerase. However, derivatives greater than 271 residues in length were sensitised to proteolysis near K271. Deletion of only 13 C-terminal amino acids impaired the response to CRP at a class I promoter whereas the complete removal of the alpha C-terminal domain did not prevent complementation of MelR-dependent PmelAB activity in the rpoA341 mutant.


CONCLUSIONS
Our results refine the C-terminal limit of the alpha assembly domain to between residues 221 and 230. The 13 extreme C-terminal amino acids are exposed in the holoenzyme and participate in the protection of an otherwise proteolytically sensitive bond near K271. Their presence is also essential for transcription activation at class I CRP-dependent promoters. The rpoA341-mediated substitution, K271E, does not define an activation contact site for MelR.
Zou
C
C
Department of Biochemistry, Queen's Medical Centre, Nottingham, UK.
Thomas
M S
MS
Keen
J
J
Glass
R E
RE
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005816:Genetic Complementation Test; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases