INVESTIGATION OF T7 RNA POLYMERASE BIOCHEMICAL AND PHYSICO-CHEMICAL PROPERTIES

Abstract

T7 RNA polymerase (T7 RNAP) is a single-subunit RNA polymerase whose activity relies on large conformational transitions during transcription. Here we employed molecular dynamics simulations to characterize how specific point mutations (S43Y, Y639V, V685A, Q744R), a double mutant (Y639V/V685A), and a quadruple mutant (S43Y/Y639V/V685A/Q744R) alter the enzyme’s dynamics at 310 K and 315 K. Wild-type and mutant T7 RNAPs (all simulated in a promoter-bound complex) were compared in terms of flexibility (root-mean-square fluctuations, RMSF), global stability (RMSD) and intramolecular interactions. All single mutations increased the flexibility of the N-terminal domain (NTD), while decreasing the flexibility of an active-site adjacent loop (residues 600–610). Notably, the quadruple mutant exhibited a lower overall RMSF at 315 K than at 310 K, indicating a gain in rigidity at elevated temperature. This anomalous thermal response suggests that the combination of mutations confers enhanced structural stability and possibly an “activated” conformational state at higher temperature. Hydrogen-bond analysis revealed that a key anchoring interaction (His411–Glu35) present in the wild type is lost in all mutants, explaining the liberated NTD movements. Our findings indicate that these mutations allosterically rewire T7 RNAP’s internal dynamics: they free the NTD and stabilize the active site region, a dual effect that may underlie known functional phenotypes (e.g. reduced termination by S43Y and expanded substrate scope by Y639V). These insights advance our understanding of structure–function relationships in T7 RNAP and inform strategies for engineering polymerases with altered stability or transcriptional properties.