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|Title:||CHARACTERIZING TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATORS IN STREPTOMYCES BACTERIA|
|Authors:||Young, Rachel A.|
|Abstract:||The final stage of transcription, termed termination, is required for proper gene expression in bacteria. However, the mechanism of transcription termination is poorly understood in the multicellular Streptomyces bacteria. Motivated by the lack of well characterized terminators, we systematically searched for intrinsic termination signals in three divergent Streptomyces species. Using a novel computational approach we identified hundreds of biologically relevant terminators in each species. Many of these terminators were found downstream of annotated genes and aligned nicely with intrinsic terminators identified by other prediction algorithms. Further in silico analyses indicated that the streptomycetes prefer to terminate transcription at non-canonical structures characterized by a long hairpin lacking a trailing U-rich tract. We prioritized three structurally diverse intrinsic terminators for in vivo analysis. Due to difficulties optimizing a fluorescent reporter assay, we have not yet been unable to measure termination efficiency in vivo. Current work is focused on developing a new reporter system that can be used to characterize putative Streptomyces terminators. In this work, we have also investigated small RNA (sRNA)-mediated regulation in Streptomyces. These bacteria encode hundreds of sRNAs, although very few have been characterized to date. As a result, we designed an affinity purification system that can be used to identify sRNA interaction partners (e.g. proteins and RNAs). Initially, we tried to implement a system using a highly structured streptavidin aptamer; however, sRNAs tagged with this aptamer were not stably expressed in vivo and did not appear to re-fold effectively in vitro. More recently, we have tagged several sRNAs with a different aptamer which binds the MS2 phage coat protein. Future experiments will focus on testing the functionality of these new RNA fusions in vivo.|
|Appears in Collections:||Open Access Dissertations and Theses|
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