Engineering of Small RNA Transcriptional Activators

RNA regulators of gene expression have become an integral component of the synthetic biology toolbox. Small RNAs (sRNAs) are an abundant and mechanistically diverse class of RNA regulator that can affect multiple aspects of gene expression. However, there are no known natural or synthetic examples of sRNAs that can directly activate transcription in vivo. Motivated by this natural gap in RNA based regulation, we re-engineered the structural logic of a naturally occurring RNA transcriptional attenuator to create sRNA transcriptional activators that function in Escherichia coli. Using two different mechanistic designs, we show that we can create trans-acting sRNAs that promote the disruption of RNA intrinsic transcriptional terminators located upstream of a target gene. Through a series of optimizations, we were able to achieve up to 90-fold activation of transcription. Moreover, by applying this strategy to a different RNA transcriptional attenuator, and to a transcriptionally repressive riboswitch, we showed that we can create additional transcriptional activators that are both orthogonal to each other, and to existing RNA transcriptional repressors. Our results demonstrate that sRNAs are capable of activating transcription, and provide a new mechanism for the regulation of gene expression.