Generation and functional studies of phage SP6 RNA polymerase mutants

Abstract

In order to identify and characterize functional amino acid residues of the SP6 RNA polymerase, the cloned gene was randomly mutagenized by polymerase chain reaction (PCR) under the conditions of reduced Taq polymerase fidelity (20 mM $MgCl_2$, 1 mM dNTP and pH 8.3 at room temperature). Through the two vector system that permits phenotypic isolation of SP6 RNA polymerase mutants with reduced enzymatic activity, 2 single and 1 multiple mutants were isolated. Then, the properties of mutant polymerases were analyzed for promoter binding activity, solubility, non-specific catalytic activity, ability to produce short and long RNAs, and recognition of T7 termination signal. The characterization of these SP6 RNA polymerase mutants showed the following; (1) Deletion of Asn559 gave rise to significant changes in enzyme properties. Asn559 may be essential for unwinding DNA helix. The Asn residue is highly conserved in phage RNA polymerases and lies near motif A found in many nucleotide polymerases. (2) In L605P, only a small amount was obtained in soluble form. Substitutions of Pro for Leu605 retained normal promoter binding activity but exhibited weak catalytic activity. (3) One multiple mutant retained normal promoter binding activity and weak catalytic activity, implying that Val872, Phe873 or Ala874 are not related to the promoter binding, and some of these residues may play roles for the catalytic activity or melting upon DNA helix.