Biochemical characterization of a family of sigma factor-associated TnpB proteins
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Liu-catieliu-phd-biology-2025-thesis .pdf
Description
Thesis PDF
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21.86 MB
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Author(s)
Liu, Catherine C.
Advisor(s)
Zhang, Feng
Date Issued
September 2025
Publisher
Massachusetts Institute of Technology
Abstract
The TnpB proteins are a widespread family of bacterial and archaeal RNA-guided enzymes thought to be the evolutionary ancestors of Cas12 endonucleases. Previous studies have identified tnpB genes associated with proteins of diverse functions, including bacterial transcriptional regulation [1-3]. In my doctoral thesis work, I characterize the biochemical properties of a family of sigma factor-associated TnpB proteins. I show that various sigma factor-associated tnpB loci encode a non-coding RNA species (ωRNA) downstream of the tnpB stop codon that binds to the purified TnpB protein. When co-expressed with their cognate ωRNAs, the sigma factor-associated TnpB proteins target and bind regions of the E. coli genome corresponding to a 4-8 nucleotide guide sequence found within the ωRNA and an additional 5’ 2-5 nucleotide target-adjacent motif (TAM). In vitro binding assays confirm that TnpB binds its double-stranded DNA target in an ωRNA- and TAM-dependent manner. Focusing on one ortholog with a well-defined TAM, I find that the TnpB protein can be partially retargeted to different regions of the E. coli genome by reprogramming its 8 nucleotide ωRNA guide sequence. I furthermore demonstrate that this TnpB co-immunoprecipitates with its associated sigma factor when both are heterologously expressed in E. coli. Collectively, these data suggest that this family of TnpBs are RNA-guided DNA-targeting proteins that may be functionally involved in transcriptional regulation through their association with bacterial sigma factors.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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