Sequence-Function Models of Regulatory RNA in E. Coli

Abstract

The tremendous progress in genome sequencing has highlighted a challenging and important problem in connecting known sequences to biological function, known or unknown. In bacteria, small regulatory RNAs (sRNAs) 50-200 nucleotides in length control the expression of genes across the gene regulatory network. They achieve this post-transcriptionally by modulating stability and translation initiation of their target mRNAs. In many cases these RNA-RNA regulatory interactions have been connected to base-pairing, and depend on specific interactions with protein co-factors. However, the sequence determinants of function for sRNAs remain poorly defined on two levels. First, parts of the nucleotide sequence have not been classified or characterized, even for well-studied sRNAs. Second, known or suspected sequence elements such as target pairing regions, which vary widely in length, and are often supported by limited evidence such as experiments from one or a few point mutants. We have sought to explore the relations between an sRNA sequence and its regulatory functions systematically. We have employed sort-seq, a parallelized approach based on sequencing of pools of cells sorted by florescence, to measure the regulatory activity of large libraries of sRNA variants generated by random mutagenesis. In addition to demonstrating and validating the method for analysis of bacterial sRNAs, we have optimized several consequential aspects of sort-seq experiments. Simulations elucidate key sources of measurement bias and error driven by cell-to-cell variability and the configuration of sorting. With informed experimental design choices, we have demonstrated extremely precise measurements where variants have sufficient coverage -- RMS replicate error less than 5% at 150X coverage. Our work has established sort-seq as a measurement platform for trans-acting RNA regulators. We have used sort-seq to explore the sequence-function relations of two sRNAs of E. coli, RyhB and DsrA. Our results define functional sequence elements for the maintenance and activity of the sRNA as well as specific interactions with different mRNA targets. The data show a relationship between thermodynamic stability of the RNA-RNA duplex between the regulator and target as well as stability of individual stem-loop structures. A survey of sort-seq measurements across a range of environmental temperatures reveals sequence elements with temperature-dependent function. We have used sequence-function models, constrained by sort-seq measurements, to uncover sequence and environmental determinants of regulatory specificity of RyhB.