Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription

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dc.contributor.advisor Gelles, Jeff
dc.contributor.author Yanding, Zhao
dc.date.accessioned 2015-09-14T18:48:07Z
dc.date.available 2015-09-14T18:48:07Z
dc.date.issued 2015
dc.identifier.uri http://hdl.handle.net/10192/31114
dc.description.abstract TALEs are sequence specific transcription activator found in phytopathogenic bacteria. Interestingly, their specificity is defined by triplet amino acid repeat sequences, which can recognize individual nucleotides in dsDNA. In crystal structures with mutants where the transcription effector domain has been deleted (TAL), we observe a fascinating structure where the TAL protein complete wraps around the dsDNA in a helical manner. There are a myriad of applications for an engineered site-specific DNA binding protein, with or without a transcription effector domain, such as genome editing and synthetic biology. Currently, however, there is no information regarding the kinetics or mechanism by which TALs bind to their respective sequences. Knowing the mechanism by which TALs find their targets and once there how long they remain bound will help in the design and application of TAL in monitoring transcription process. By engineering a SNAP-tagged TAL protein mutant, which can be fluorescently labeled with no transcriptional effector, used in co-localization of single molecules (COSMOS) experiment, we revealed that TAL, verified to be a highly specific target DNA binding protein, formed a very stable complex with target DNA. Moreover, We used this result as a springboard to incorporate fluorescent TAL with RNA polymerase (Holoenzyme) in single molecule experiments and found transcription process was blocked by TAL when TAL-DNA complex was formed. During the transcription, the RNA polymerase was stuck on the DNA with transcribed RNA fragments and cannot overpass the TAL protein.
dc.description.sponsorship Brandeis University, Graduate School of Arts and Sciences
dc.format.mimetype application/pdf
dc.language English
dc.language.iso eng
dc.publisher Brandeis University
dc.relation.ispartofseries Brandeis University Theses and Dissertations
dc.rights Copyright by Yanding Zhao 2015
dc.subject Fluorescent
dc.subject TAL protein
dc.subject single-molecule transcription
dc.title Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription
dc.type Thesis
dc.contributor.department Graduate Program in Molecular and Cell Biology
dc.degree.name MS
dc.degree.level Masters
dc.degree.discipline Molecular and Cell Biology
dc.degree.grantor Brandeis University, Graduate School of Arts and Sciences
dc.description.esploro 2

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