Studies of Transcription Fidelity by T7 RNA Polymerase, and the Potential Applications of T7 RNA Polymerase as a Molecular Motor.

Abstract

This work comprises two studies involving bacteriophage T7 RNA polymerase (RNAP). In the first study, we explored the potential applications of T7 RNAP as a tightly regulated molecular motor in nanotechnology. In the second study, we investigated the mechanism by which T7 RNAP selects for correct nucleotide substrates during transcription. RNAP converts the chemical energy stored in ribonucleoside triphosphates (NTPs) into the mechanical work of transcription. Previous studies have shown that RNAP can exert forces up to 30 piconewtons (pN) as it moves along the DNA while copying the information in the DNA template into RNA. The forward motion of RNAP is dependent on the availability of the next incoming (correct) NTP that is encoded by the template strand of the DNA. Withholding a required NTP results in the formation of a stable halted elongation complex (EC) in which the enzyme remains bound to the DNA until transcription is resumed by addition of substrate. Immobilizing RNAP or the DNA template to which it is bound to a solid surface confers the ability to add and wash away different mixtures of substrates for individual transcription steps. In this way RNAP can be incrementally positioned along DNA with intervals as small as 1 base-pair (bp) or 0.34 nanometer (nm). To investigate the potential application of RNAP in nanotechnology a modified version of T7 RNAP that contains specific ligand binding domains fused to the amino-terminus of the enzyme was utilized to facilitate capture, controlled movement, and positioning of other biomolecules, and to construct and manipulate simple nanodevices. The ability to harness the linear and rotary forces exerted by single molecules of T7 RNAP was also examined. In the former case, fluorescence microscopy was used to observe unidirectional linear translocation of a nanodot cross-linked to T7 RNAP along immobilized T7 DNA. In the latter case, the ability of immobilized T7 RNAP to rotate a reporter bead, which was tethered to the downstream end of the DNA, was demonstrated using a magnetic tweezers apparatus. In the second part of this work, the fidelity of transcription by T7 RNAP was investigated. Sequence and structural comparisons indicate that T7 RNAP is related to members of a superfamily of nucleotide polymerases that include DNA directed DNA polymerases (DNAPs) of the pol I-type, mitochondrial RNA polymerases (mt RNAP), and reverse transcriptases (RT). These enzymes share a conserved structure around the catalytic site that resembles a partially closed right hand and includes fingers, palm and thumb subdomains. Structural studies indicate that T7 RNAP and bacterial DNAP I possess a similar catalytic pathway in which the active site undergoes a transition from a catalytically inactive “open” conformation to an active “closed” conformation following substrate binding. Kinetic and structural data indicate that the open to closed conformational change acts as a fidelity checkpoint mechanism, whereby a tight-fit geometry of the active site (in the closed conformation) precludes the acceptance of non-Watson-Crick base pairs. It has also been suggested that this mechanism might contribute to frameshift fidelity by regulating the transfer of the template DNA base (TSn) from a pre-insertion site (in the open configuration) to the insertion site (active site) in the closed configuration. Genetic analyses of T7 RNAP and DNAP I indicate that residues involved in fidelity are predominately located within the pre-insertion and insertion sites, respectively. Thus, it has been suggested that T7 RNAP may select the correct nucleotide in the pre-insertion site, prior to catalysis. Whether a similar mechanism might contribute to fidelity of DNAP I remains an open question. In a manner similar to that used in studies of DNAP to measure fidelity, primer/template (p/t) assemblies were used to investigate misincorporation by T7 RNAP. It was found that T7 RNAP can generate errors by a novel mechanism involving template strand re-alignment. Here, the template base immediately downstream from the active site (the TSn+1 base) is utilized during two consecutive nucleotide addition cycles. In the first cycle, the NTP complementary to TSn+1 (the +1 NTP) is incorporated due to transient formation of a –1 frameshift in which the proper template base (TSn) is presumably “flipped out” of the active site. In the second cycle, the template is re-aligned and the mismatched p/t is rapidly extended resulting in a substitution in the RNA. Changes in the organization of the transcription bubble and alanine substitution of residues Y639 and F644 near the active site were found to affect the rate of substitution errors. Lastly, T7 RNAP was found to generate identical substitution errors on templates in which transcription was initiated from a promoter suggesting that this phenomenon might be relevant to native transcription complexes within the cell.