Small ORFs

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Identification and characterization of small ORFs[unreadable] [unreadable] In our genome-wide screens for small RNAs, we found that a number of short RNAs actually encode small proteins. The correct annotation of the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short ORFs have been confirmed to correspond to synthesized proteins. Although small proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses. Thus we established a project to identify E. coli proteins of less than 50 amino acids and elucidate their functions using many of the approaches the group has used to characterize the functions of small, noncoding RNAs. [unreadable] [unreadable] We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16-50 amino acids, in the intergenic regions of the Escherichia coli genome. We tested expression of these predicted as well as previously annotated small proteins by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16-50 amino acids are synthesized. Remarkably more than half of the newly discovered proteins are predicted to be single transmembrane proteins, nine of which we show co-fractionate with cell membranes. Systematic screens for growth conditions that lead to increased expression and for phenotypes associated with null mutations are beginning to provide insights into the physiological roles of these small proteins.[unreadable] [unreadable] Characterization of the OxyR and Fur transcription regulators[unreadable] [unreadable] Previously, a major focus of the laboratory was the characterization of the OxyR transcription regulator, particularly its sensitivity to oxidation and its binding to DNA. We concluded this project with a study of OxyR mutants to define a region where OxyR contacts RNA polymerase. In collaboration with Thomas Schneider, we also completed a computational analysis of DNA sites bound by the iron repressor protein Fur.