Probing the nucleotide binding sites in T7 RNA polymerase using cibacron blue

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. In this work we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, and its effect on the function of the enzyme. T7 RNAP binds to the dye in a bi-phasic manner. The first phase of the binding is characterized by a high affinity (Kd in the nanomolar range) and reversible inactivation of the enzyme. The second binding site is the common substrate binding site. The association of the dye with T7 RNAP is a good model to understand the physiological significance of a high affinity binding of the initiating nucleotide, GTP, earlier reported from our laboratory. The results will be discussed to understand the role of the high affinity GTP binding.     

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.     

Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation

We have characterized the misincorporation properties of wild-type (wt) T7 RNAP and of 45 T7RNAP point mutants. The wt enzyme selects strongly against incorporation of an incorrect nucleotide. From the measured rates of misincorporation, an average error frequency of 1 in 2 x 10(4) is estimated. RNAs bearing 3'-mismatches are extended more slowly than correctly paired 3'-termini, and mismatches one or two bases away from the RNA 3'-end can also slow extension severely even when the 3'-base is correctly paired. Though it has been reported that T7RNAP has a 3' --> 5' nuclease activity, we were unable to detect any endogenous T7RNAP RNase activity in elongation complexes. Pyrophosphorolysis was detected but does not appear to contribute to proofreading. Therefore, unlike other RNAPs, T7RNAP fidelity appears to depend entirely on discrimination against incorporation of the incorrect nucleotide and not on post-misincorporation proofreading. Alanine substitution of the H784 side chain, which interacts with the 3' RNA.template base pair, increases both misincorporation and mismatch extension, while substitutions at G640, F644, and G645 increase misincorporation, but not mismatch extension. The latter three amino acids are in a part of the RNAP which interacts with the templating base and with the base immediately 5' to the templating base. Mutation of these amino acids not only increases misincorporation, but also eliminates pausing during promoter clearance. The effects of these mutations and the interactions observed in a crystal structure of a transcribing complex indicate that these mutations disrupt interactions which limit misincorporation rates by stabilizing the catalytically incompetent open conformation of the RNAP.     

Substrate properties of C′-methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N-type conformation.