Quantitative effect of target translation on small RNA efficacy reveals a novel mode of interaction

Small regulatory RNAs (sRNAs) in bacteria regulate many important cellular activities under normal conditions and in response to stress. Many sRNAs bind to the mRNA targets at or near the 5′ untranslated region (UTR) resulting in translation inhibition and accelerated degradation. Often the sRNA-binding site is adjacent to or overlapping with the ribosomal binding site (RBS), suggesting a possible interplay between sRNA and ribosome binding. Here we combine quantitative experiments with mathematical modeling to reveal novel features of the interaction between small RNAs and the translation machinery at the 5′UTR of a target mRNA. By measuring the response of a library of reporter targets with varied RBSs, we find that increasing translation rate can lead to increased repression. Quantitative analysis of these data suggests a recruitment model, where bound ribosomes facilitate binding of the sRNA. We experimentally verified predictions of this model for the cell-to-cell variability of target expression. Our findings offer a framework for understanding sRNA silencing in the context of bacterial physiology

Genomic mining of prokaryotic repressors for orthogonal logic gates

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10[superscript 54] circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.United States. Air Force Office of Scientific Research (Award FA9550-11-C-0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a)United States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4016)United States. Office of Naval Research (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210