Abstract ▼ AI-Helper

In order to identify and characterize functional amino acid residues that are close to the N-terminus of the bacteriophage SP6 RNA polymerase, the cloned gene was randomly mutagenized by polymerase chain reaction under the conditions of reduced Taq polymerase fidelity. Deoxy-ITP (200 $\mu$M) was add...

In order to identify and characterize functional amino acid residues that are close to the N-terminus of the bacteriophage SP6 RNA polymerase, the cloned gene was randomly mutagenized by polymerase chain reaction under the conditions of reduced Taq polymerase fidelity. Deoxy-ITP (200 $\mu$M) was added to a mixture of deoxynucleotides where only one of the nucleotides was at 15 M and the rest at 200 M. Or, high concentrations of Mg2+ (20 mM) and nucleotides (1 mM) were used at a high pH (8.3). Through a two-vector system that permits phenotypic isolation of the polymerase mutants with reduced enzymatic activity in vivo, 5 single- and 3 multiple mutants were isolated as well as many truncated mutants. The isolated single mutants of C80T, D117V, S138L, Q179P and K231I, indicate the importance of the residues at 80, 117, 138, 179 and 231. The double mutants of L139P+T144A and I182F+T184A, and the triple mutant of L190P+Y196C+S238G indicate the potentially essential residues. The S138L mutant has normal promoter binding activity but low catalytic activity. The D117V losts not only promoter binding but catalytic activity. This D117 is located conserved region of domain 1(McAllister and Raskin, 1993), this means it is potentially important residue of phage RNA polymerase. The C80T has low promoter binding but surprisingly high catalytic activity. The C80T mutant has more nonspecific DNA(for example poly(dI,dC)) binding activity. The K231I has little low catalytic activity. The Q179P is overall low activity. The L139P+T144A binds to promoter weekly, have low catalytic activity. The I182F+T184A and the L190P+Y196C+S238G have low overall transcription activity. Deletion mutants include very low activity of transcription.