title: Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase.
abstract: We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long beta subunit is alternatively cleaved at Arg903 or Lys909. The cleavage occurs adjacent to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue 1000), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Arg81 of the beta' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts.
authors.affiliation: Department of Microbiology, Columbia University College of Physicians and Surgeons, New York 10032.
authors.forename: S
authors.initials: S
authors.lastname: Borukhov
authors.forename: K
authors.initials: K
authors.lastname: Severinov
authors.forename: M
authors.initials: M
authors.lastname: Kashlev
authors.forename: A
authors.initials: A
authors.lastname: Lebedev
authors.forename: I
authors.initials: I
authors.lastname: Bass
authors.forename: G C
authors.initials: GC
authors.lastname: Rowland
authors.forename: P P
authors.initials: PP
authors.lastname: Lim
authors.forename: R E
authors.initials: RE
authors.lastname: Glass
authors.forename: V
authors.initials: V
authors.lastname: Nikiforov
authors.forename: A
authors.initials: A
authors.lastname: Goldfarb
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin