Biology subjects

Meger, A. T.

Publications and source records attributed to Meger, A. T..

3 recordsLinked to original sources

bioRxiv2022

Computation-guided redesign of promoter specificity of a bacterial RNA polymerase

The ability to regulate genetic circuits and metabolic pathways is central to cellular control. The existing toolkit is predominantly comprised of local transcription regulators that are unsuitable for exerting control at a global genome-wide scale. Bacterial sigma factors are ideal global regulators as together they direct the RNA polymerase to thousands of transcription sites. Here, we redesigned the promoter specificity of the E. coli housekeeping sigma factor, sigma-70, toward five orthogonal promoter targets not recognized by the native sigma-70. These orthogonal sigma-70 factors were developed by screening a pooled library of computationally designed variants of the -35 DNA recognition helix, each tailored to a specific target promoter. In the redesigned sigma factors new target-specific interactions facilitate new promoter recognition. Activity of the top performing redesigned sigma-70s varied across the promoter targets and ranged from 17% to 77% of native sigma-70 on its canonical active promoter. These orthogonal sigma factors represent a new suite of regulators for global transcriptional control.

synthetic biology↗

bioRxiv2022

The rugged DNA-binding sequence-fitness landscape of the LacI/GalR Family is a product of asymmetry in the operator:repressor complex

How a proteins function influences the shape of its fitness landscape, smooth or rugged, is a fundamental question in evolutionary biochemistry. Smooth landscapes arise when incremental mutational steps lead to a progressive change in function, as commonly seen in enzymes and binding proteins. On the other hand, rugged landscapes are poorly understood because of the inherent unpredictability of how sequence changes affect function. Here, we experimentally characterize the entire sequence phylogeny, comprising 1158 extant and ancestral sequences, of the DNA-binding domain (DBD) of the LacI/GalR transcriptional repressor family. Our analysis revealed an extremely rugged landscape with rapid switching of specificity even between adjacent nodes. Further, the ruggedness arises due to the necessity of the repressor to simultaneously evolve specificity for asymmetric operators and disfavors potentially adverse regulatory crosstalk. Our study provides fundamental insight into evolutionary, molecular, and biophysical rules of genetic regulation through the lens of fitness landscapes.

biochemistry↗

bioRxiv2019

Computation-guided optimization of split protein systems

Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biochemical systems. However, split proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants--which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic activity--to identify an optimal functional window. We validate our method by solving two distinct split protein design challenges, generating both broad insights and new technology platforms. This method will streamline the generation and use of split protein systems for diverse applications.

synthetic biology↗