Disruption of Escherichia coli hepA, an RNA polymerase-associated protein, causes UV sensitivity.
During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide. Here, we report the identification of the 110-kDa protein as the product of the hepA gene, a member of the SNF2 family of putative helicases. We have cloned the hepA gene and overexpressed and purified the HepA protein. We show in vitro that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA binds core RNAP competitively with the promoter specificity sigma70 subunit with a 1:1 stoichiometry and a dissociation constant (Kd) of 75 nM. An E. coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.
Muzzin
O
O
Rockefeller University, New York, New York 10021, USA.
Campbell
E A
EA
Xia
L
L
Severinova
E
E
Darst
S A
SA
Severinov
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D017421:Sequence Analysis; D012808:Sigma Factor / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074434:RapA protein, E coli; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases