Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit.
We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. Trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of sigma 70.sigma 70(2) bound core RNA polymerase competitively with intact sigma 70. In contrast to sigma 70(2) alone, the RNA polymerase holoenzyme formed with sigma 70(2) specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box). Sigma 70(2) also forms crystals that are suitable for X-ray analysis. Sigma 70(3-4) bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on sigma 70 was further localized to within sigma 70[551-608], comprising sigma conserved region 4.2.
Severinova
E
E
Laboratory of Molecular Biophysics, Rockefeller University, New York, NY 10021, USA.
Severinov
K
K
Fenyö
D
D
Marr
M
M
Brody
E N
EN
Roberts
J W
JW
Chait
B T
BT
Darst
S A
SA
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000378:metabolism; D001667:Binding, Competitive; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014357:Trypsin / Q000737:chemistry; D014764:Viral Proteins / Q000378:metabolism
C078876:AsiA protein, Enterobacteria phage T4; D011994:Recombinant Proteins; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin