Biology subjects

Miller, R. C.

Publications and source records attributed to Miller, R. C..

2 recordsLinked to original sources

bioRxiv2025

Single-molecule microscopy reveals that TFIIE subunits dynamically interact with preinitiation complexes in a manner controlled by TFIIH

Transcription by RNA polymerase II (Pol II) requires general transcription factors that bind with Pol II at the promoters of protein-coding genes to form preinitiation complexes (PICs). Among these is TFIIE, which recruits TFIIH to the PIC and stimulates the kinase and translocase activities of TFIIH, thereby regulating the fate of formed PICs. In this study, we used a purified reconstituted human Pol II transcription system and single molecule total internal reflection fluorescence (smTIRF) microscopy to monitor TFIIE binding dynamics in PICs under different conditions in real time. We observed highly dynamic interactions of the two subunits of TFIIE (TFIIE and TFIIE{beta}) with PICs. Measurement of rate constants for on/off binding of each subunit suggest they behave asynchronously. TFIIH exclusion increased the rates of association and dissociation for both subunits, with the strongest effect on TFIIE. Despite stabilization of TFIIE by TFIIH the TFIIE subunits remain dynamic in PICs. Additionally, two disease-related TFIIE{beta} point mutations destabilized TFIIE{beta} and altered its kinetic behaviors within PICs. Our results contribute to an emerging model that PICs are not static assemblies and highlight important connections between the structural arrangement and kinetic behaviors of GTFs in PICs.

biochemistry↗

bioRxiv2024

Mechanism of Nucleotide-Dependent Allosteric Regulation in Escherichia coli Aspartate Transcarbamoylase

Aspartate transcarbamoylase (ATCase) from Escherichia coli catalyzes a key step in pyrimidine nucleotide biosynthesis and has long served as a model for allosteric regulation. Despite decades of study, how nucleotide binding at distant regulatory sites controls cooperativity between active sites remained unresolved. Here we show that ATCase does not simply interconvert between two conformations, as traditionally depicted, but instead samples a continuum of conformations that tune enzyme cooperativity. Using complementary cryo-electron microscopy, small-angle X-ray scattering, and crystallography under conditions that ensure full assembly of the allosteric sites, we show that ATCase behaves like a flexible balloon whose global "breathing" motions directly regulate activity: compression enforces high cooperativity, inhibiting the enzyme, whereas expansion relieves this cooperativity and activates the enzyme. We further show that all four ribonucleoside triphosphates act in symmetric pairs to tune this motion, with the pyrimidines CTP and UTP compressing the enzyme to limit further pyrimidine production, and the purines ATP and GTP expanding it to balance pyrimidine and purine pools. Together, these findings uncover a dynamic breathing mechanism for long-range allosteric communication in ATCase.

biochemistry↗