Dataset Information

Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors.

ABSTRACT: T7 RNA polymerase (RNAP) is a powerful protein scaffold for the construction of synthetic biology tools and biosensors. However, both T7 RNAP and its split variants are intolerant to C-terminal modifications or fusions, thus placing a key limitation on their engineering and deployment. Here, we use rapid continuous-evolution approaches to evolve both full-length and split T7 RNAP variants that tolerate modified C?termini and fusions to entire other proteins. Moreover, we show that the evolved split C-terminal RNAP variants can function as small-molecule biosensors, even in the context of large C-terminal fusions. This work provides a panel of modified RNAP variants with robust activity and tolerance to C-terminal fusions, and provides insights into the biophysical requirements of the C-terminal carboxylic acid functional group of T7 RNAP.

SUBMITTER: Pu J

PROVIDER: S-EPMC6570552 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors.

Pu Jinyue J Disare Michael M Dickinson Bryan C BC

Chembiochem : a European journal of chemical biology 20190417 12

T7 RNA polymerase (RNAP) is a powerful protein scaffold for the construction of synthetic biology tools and biosensors. However, both T7 RNAP and its split variants are intolerant to C-terminal modifications or fusions, thus placing a key limitation on their engineering and deployment. Here, we use rapid continuous-evolution approaches to evolve both full-length and split T7 RNAP variants that tolerate modified C termini and fusions to entire other proteins. Moreover, we show that the evolved sp ...[more]

PMID: 30694596

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Project description:With a simple tandem iterated sequence, the carboxyl terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II) serves as the central coordinator of mRNA synthesis by harmonizing a diversity of sequential interactions with transcription and processing factors. Despite intense research interest, many key questions regarding functional and evolutionary constraints on the CTD remain unanswered; for example, what selects for the canonical heptad sequence, its tandem array across organismal diversity, and constant CTD length within given species and finally and how a sequence-identical, repetitive structure can orchestrate a diversity of simultaneous and sequential, stage-dependent interactions with both modifying enzymes and binding partners? Here we examine comparative sequence evolution of 58 RNAP II CTDs from diverse taxa representing all six major eukaryotic supergroups and employ integrated evolutionary genetic, biochemical, and biophysical analyses of the yeast CTD to further clarify how this repetitive sequence must be organized for optimal RNAP II function. We find that the CTD is composed of indivisible and independent functional units that span diheptapeptides and not only a flexible conformation around each unit but also an elastic overall structure is required. More remarkably, optimal CTD function always is achieved at approximately wild-type CTD length rather than number of functional units, regardless of the characteristics of the sequence present. Our combined observations lead us to advance an updated CTD working model, in which functional, and therefore, evolutionary constraints require a flexible CTD conformation determined by the CTD sequence and tandem register to accommodate the diversity of CTD-protein interactions and a specific CTD length rather than number of functional units to correctly order and organize global CTD-protein interactions. Patterns of conservation of these features across evolutionary diversity have important implications for comparative RNAP II function in eukaryotes and can more clearly direct specific research on CTD function in currently understudied organisms.

Dataset Information

Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors.

Publications

Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors.

Similar Datasets

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