Borukhov S, Severinov K, Kashlev M, Lebedev A, Bass I, Rowland G C, Lim P P, Glass R E, Nikiforov V, Goldfarb A
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York 10032.
J Biol Chem. 1991 Dec 15;266(35):23921-6.
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.
我们已经绘制了大肠杆菌RNA聚合酶分子中在有限蛋白水解条件下易受胰蛋白酶攻击的主要位点。1342个氨基酸长的β亚基在Arg903或Lys909处被选择性切割。切割发生在一个可有可无的结构域（残基940 - 1040）附近，该结构域在叶绿体、真核生物和古细菌的同源RNA聚合酶亚基中不存在。在大肠杆菌中，该区域可通过基因缺失和插入而被破坏，而不会丧失RNA聚合酶的功能。在该区域插入127个氨基酸会引入一个新的对胰蛋白酶蛋白水解高度不稳定的位点。这个可有可无的结构域携带单克隆抗体PYN - 6的表位（靠近残基1000），可用于将催化活性酶固定在固体支持物上。我们还报告了在β'亚基的Arg81处鉴定出一个二级胰蛋白酶切割位点，该位点位于原核生物和叶绿体中保守的一个假定锌结合结构域内。
