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dc.contributor.advisorGaudet, Rachelle
dc.contributor.authorSalguero, Carolina
dc.date.accessioned2019-05-20T10:22:26Z
dc.date.created2017-05
dc.date.issued2017-05-18
dc.date.submitted2017
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:40046412*
dc.description.abstractStable RNA structures interact with proteins at a frequency that allows for regulation of cellular and viral processes. Using a combination of Nuclear Magnetic Resonance (NMR), Small Angle X-Ray Scattering, Mass-Spectrometry and translational assays, I investigated two viral mRNA signals to understand what determines the frequency of recoding events that allows for fine-tuning of gene expression at the translational level. Here, I show that mutating loop residues that have the potential of forming long-range interactions with the major groove, and changing the pH of the translational mixture increases recoding efficiency of the SARS-Coronavirus pseudoknot (SARS-PK) in-vitro. In addition, I show that the SARS-PK is slightly flexible and the particular geometry of its junction induces a slight bent, which is sensitive to pH. Exploring the flexibility and ability of recoding signals to sample multiple conformations, I also present a minimal sequence required for optimal recoding efficiency in HIV-1, as well as, a set of mutations that partition the RNA into two conformations in-vitro and render the HIV-1 signal hyperactive in-vivo. Collectively, my results support a model where the SARS and HIV-1 recoding signals sample multiple conformations, but only one of them, the active signal, induces a fraction of the ribosomes to recode. In the second chapter, I investigated structural features of the 7SK- snRNA, which regulates the availability of the elongation factor b (p-TEFb) required for productive transcriptional elongation of HIV-1 and other cellular genes. I present a high-resolution structure of the first stem-loop of 7SK-snRNA (7SK-SL1) and show that one of its multiple pyrimidine bulges closely resembles the viral TAR-RNA. To understand how the RNA binding domain of the viral Tat protein binds 7SK-SL1 first and the viral TAR-RNA later, I also present a high-resolution structure of 7SK-SL1 in complex with the arginine-rich motif of Tat, and a binding comparison to the arginine-rich motifs of the cellular HEXIM and Brd4 by isothermal titration calorimetry (ITC) and NMR. Collectively, my results show structural mimicry between 7SK- snRNA and TAR-RNA, and that Tat’s arginine-rich motif displays structural advantages that may allow it to bind 7SK-SL1 first.
dc.description.sponsorshipBiology, Molecular and Cellular
dc.format.mimetypeapplication/pdf
dc.language.isoen
dash.licenseLAA
dc.subjectBiology, Molecular
dc.subjectChemistry, Biochemistry
dc.titleStructural and Biochemical Studies of RNA Domains Involved in Regulation of Transcriptional Elongation of the HIV-1 retrovirus and Translational Recoding of the SARS Coronavirus
dc.typeThesis or Dissertation
dash.depositing.authorSalguero, Carolina
dc.date.available2019-05-20T10:22:26Z
thesis.degree.date2017
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
dc.contributor.committeeMemberLosick, Richard
dc.contributor.committeeMemberSummers, Mike
dc.type.materialtext
thesis.degree.departmentBiology, Molecular and Cellular
dash.identifier.vireohttp://etds.lib.harvard.edu/gsas/admin/view/1405
dc.description.keywordsRNA structure; Pseudoknot; Translational Recoding; Transcriptional Elongation
dash.author.emailcsalguerob@gmail.com


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