Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase
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 (residue...
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Veröffentlicht in: | The Journal of biological chemistry 1991-12, Vol.266 (35), p.23921-23926 |
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Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(18)54372-6 |