DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.     

Conversion of Escherichia coli RNA polymerase to a template independent enzyme.

Preparations of RNA polymerase (E.C.2.7.7.6) from uninfected Escherichia coli, T4 infected Escherichia coli, and Acinetobacter calcoaceticus when centrifuged in sucrose gradients in the absence of magnesium ions gave rise to five peaks, all of which were able to form polymers from ribonucleoside 5'-triphosphates in the absence of template or primer. All of the peaks obtained from the Escherichia coli enzyme appeared to contain the subunit alpha and beta and, in addition, polypeptides which appeared to be derived from the subunit beta.     

Promoter search by Escherichia coli RNA polymerase on a circular DNA template

Using the rapid-mixing/photocross-linking technique developed in our laboratory, we have investigated the kinetics of interaction between Escherichia coli RNA polymerase and pAR1319, a recombinant plasmid DNA containing the bacteriophage T7 A2 early promoter. By monitoring the time-dependent density of bound RNA polymerase along the relaxed circular DNA molecule using this technique, we have been able to demonstrate kinetic evidence for linear diffusion of RNA polymerase along DNA in a different system from that previously described (Park, C. S., Hillel, Z., and Wu, C.-W. (1982) J. Biol. Chem. 251, 6950-6956). The nonspecific association rate constant kon was measured to be 7.7 x 10(4) M-1 s-1 at a DNA chain concentration of 22.4 nM. By taking advantage of the fact that rapid mixing displaces bound protein molecules from DNA, but leaves them within the domain of the DNA, the rate of intradomain binding of RNA polymerase to pAR1319 DNA was determined to be 8.2 s-1. Since the plasmid is described by a radius of gyration of 0.22 microns, the intradomain concentration of base pairs could be calculated. Using this concentration (180 microM), the rate constant for intradomain nonspecific association of RNA polymerase to pAR1319 DNA was estimated to be 4.6 x 10(4) M-1 s-1. In addition, a mathematical model has been used to fit the other two important rate constants to the experimental data: koff, which describes the dissociation of RNA polymerase from nonspecific binding sites, and D1, the one-dimensional diffusion coefficient of the enzyme along the DNA molecule. In this model, the circular DNA molecule is described as a ring of interconnected binding sites which together comprise a DNA "domain." RNA polymerase, which enters the domain via three-dimensional diffusion and binds to each site, is allowed to diffuse linearly between adjacent sites and three-dimensionally on and off the DNA molecule. The rate equations for the time-dependent occupancy of each site by RNA polymerase could be written, based on general principles. By solving the resulting family of differential equations, koff and D1 were determined to be 0.3 s-1 and 1.5 x 10(-9) cm2 s-1, respectively.     

RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.