Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.

25Splitting bioactive proteins, such as enzymes or fluorescent reporters, into conditionally reconstituting 26 fragments is a powerful strategy for building tools to study and control biochemical systems. However, split 27 proteins often exhibit a high propensity to reconstitute even in the absence of the conditional trigger, which 28 limits their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or 29 often ineffective. Here, we report a computational design-driven strategy that is grounded in fundamental 30 protein biophysics and which guides the experimental evaluation of a focused, sparse set of mutants-31 which vary in the degree of interfacial destabilization while preserving features such as stability and catalytic 32 activity-to identify an optimal functional window. We validate our method by solving two distinct split 33 protein design challenges, generating both broad insights and new technology platforms. This method will 34 streamline the generation and use of split protein systems for diverse applications. 35 36 KEYWORDS: synthetic biology, split proteins, computational protein design, protein engineering 37 38

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The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that at the end of RNA transcripts, tracts comprised of multiple uracils are required for Pol III termination, whether upstream RNA secondary structure in the nascent transcript is necessary for robust transcriptional termination is still subject to debate. We sought to address this directly through the development of an in cellulo Pol III transcription termination assay using a synthetic biology approach. Specifically, we utilized the recently developed Tornado expression system and a stabilized Corn RNA aptamer to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. We found that in the absence of predicted secondary structure, only poly-U tracts longer than then the average length found in the human genome (4–5 nucleotides), efficiently terminate Pol III transcription. We found that shorter poly-U tracts could induce termination when placed in proximity to secondary structural elements, while secondary structure by itself was not sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.