(646d) Discovery and Design of Novel Regulatory Noncoding RNA in Bacteria

The work here describes the identification of new regulatory noncoding RNAs in bacteria and their potential use in synthetic biology applications. Produced during futile attempts by the RNA polymerases to clear the promoter region and transition to transcriptional elongation phase, the synthesis of such “abortive” RNAs have been known for decades in all kingdoms of life. However, until the recent identification of abortive RNA in the bacteriophage T7 with transcriptional antitermination function, they had been considered only as “junk” RNAs. Functional abortive RNAs have not, however, been identified to exist endogenously in more complex biological systems such as bacteria. Using an in silico predictive approach, we evaluated whether these abortive RNAs might be able to serve biological functions in E. coli. Abortive RNAs generated from 3,780 transcriptional units (obtained from RegulonDB) were used as query sequences within their respective transcription units to search for possible binding sites. Sites that fell within known regulatory features, based upon data obtained from RegulonDB and WebGeSTerDB, were then ranked based upon the standard free energy of annealing (reported by UNAfold) of the transcript to the binding site. Matches with a sufficiently high standard free energy of annealing were then explored further as likely sites for abortive RNA-mediated regulation. We will discuss one such result – the uncovering of a potentially novel abortive RNA-mediated regulation of an alcohol dehydrogenase in E. coli. Future experimental validation of this and other loci may reveal the pervasive physiological role of this new class of noncoding RNAs in E. coli. We also discuss the potential application of using such functional abortive RNAs in a design-based approach to regulating gene expression in synthetic systems.