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Control over the timing, location and level of protein activity in vivo is crucial to understanding biological function. Living systems are able to respond to external and internal stimuli rapidly and in a graded fashion by maintaining a pool of proteins whose activities are altered through post-translational modifications. Here we show that the process of protein trans-splicing can be used to modulate enzymatic activity both in cultured cells and in Drosophila melanogaster. We used an optimized conditional protein splicing system to rapidly trigger the in vivo ligation of two inactive fragments of firefly luciferase in a tunable manner. This technique provides a means of controlling enzymatic function with greater speed and precision than with standard genetic techniques and is a useful tool for probing biological processes.

SUMMARY The σ factors are the key regulators of bacterial transcription initiation. Through direct read-out of promoter DNA sequence, they recruit the core RNA polymerase to sites of initiation, thereby dictating the RNA polymerase promoter-specificity. The group 1 σ factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are auto-regulated by an N-terminal sequence known as σ1.1. Here we report the solution structure of Thermatoga maritima σA σ1.1. We additionally demonstrate by using chemical crosslinking strategies that σ1.1 is in close proximity to the promoter recognition domains of σA. We therefore propose that σ1.1 auto-inhibits promoter DNA binding of free σA by stabilizing a compact organization of the σ factor domains that is unable to bind DNA.

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A complex interplay between DNA, histones, and chromatin associated proteins coordinates the structure and function of the eukaryotic genome. To help accomplish this task, extensive posttranslational modifications of the core histone proteins have evolved. One of the less elucidated modifications, monoubiquitylation of H2B (uH2B), has been implicated in transcription elongation and histone methylation of H3 K4 and K79. Characterization of the role of uH2B in these processes requires the isolation or generation of homogenously ubiquitylated H2B. To this end, we have developed a traceless semisynthetic strategy to generate uH2B. We employ two expressed protein ligation (EPL) reactions and two methods for ligation site removal to synthesize native uH2B. The resulting uH2B was reconstituted into core histone octamers and nucleosome arrays were then generated for biochemical characterization. Controlling protein function through posttranslational manipulations has emerged as an attractive complimentary technology to existing genetic systems. We have developed a new posttranslational, small molecule‐mediated, technology for the manipulation of protein function. This system, termed SURF (Split‐Ubiquitin for the Rescue of Function), places the complementation of ubiquitin under the control of a small molecule. Before complementation a protein of interest is targeted for destruction by the proteasome through the introduction of an N‐terminal degron. Small molecule induced dimerization results in ubiquitin complementation and folding followed by cleavage of the protein of interest, thereby releasing it from the degron and rescuing its function. This system has been successfully applied to activate several classes of proteins in living cells. This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained.