{'fraction_non_alphanumeric': 0.013847080072245636, 'fraction_numerical': 0.001806140878988561, 'mean_word_length': 5.783673469387755, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Engineering mRNA structural regulation of transcription using an RNA‐sensing riboregulator

The control of gene expression lies at the heart of every genetic engineering enterprise. However, current strategies to manipulate gene expression are dominated by inefficient trial and error screening approaches. Here we experimentally investigate an RNA‐based transcription attenuation mechanism in E. coli with the aim of engineering a family of mutually orthogonal riboregulators that form a platform for the forward design of expression architectures. We focus on the antisense‐RNA‐mediated transcription regulator of S. aureus plasmid pT181 as the basis for this family of control units. Comparative sequence information from similar RNA‐mediated translation mechanisms suggest hypotheses about the detailed binding pathway between antisense and sense RNAs that enable us to investigate the mechanistic origin of specificity in this system with mutational analysis. We demonstrate how these investigations allow us to change specificity in this system, and construct orthogonally‐acting transcription regulators. Furthermore, we show how these transcriptional regulators can be composed in series on the same transcript to give increased cooperativity and the ability to integrate signals on a single transcriptional locus. Finally, we demonstrate how they can be chained together to create a two‐stage RNA‐mediated transcriptional cascade that opens the door for creating general feedback loops with this system. This work is funded as a part of the NSF Synthetic Biology Engineering Resource Center grant and the Miller Institute for Basic Research in Science.