Single Molecule FRET Studies of Reverse Transcription and Chromatin Remodeling
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CitationHarada, Bryan T. 2015. Single Molecule FRET Studies of Reverse Transcription and Chromatin Remodeling. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractThe measurement of Förster resonance energy transfer (FRET) at the single-molecule level provides a powerful method for monitoring the structural dynamics of biomolecular systems in real time. These single-molecule FRET (smFRET) assays enable the characterization of the transient intermediates that form during enzymatic processes, providing information about the mechanism and regulation of the enzymes involved. In this dissertation, I develop and use smFRET assays to study two processes driven by motor proteins—reverse transcription and chromatin remodeling—and reveal novel features of their mechanism and regulation.
Reverse transcription of the human immunodeficiency virus genome initiates from a cellular tRNA primer that is bound to a specific sequence on the viral RNA (vRNA). During initiation, reverse transcriptase (RT) exhibits a slow mode of synthesis characterized by pauses at specific locations, and RT transitions to a faster mode of synthesis after the extension of the tRNA primer by six nucleotides. By using smFRET to examine how RT interacts with the tRNA-vRNA substrate, we found that RT binds to its substrate in either an active or inactive orientation and samples the two orientations during a single binding event. The equilibrium between these two orientations is a major factor influencing the activity and pausing of the enzyme, and a specific RNA secondary structure in the vRNA substrate modulates the binding mode of RT, determining the locations of the pauses and the transition to the faster mode of synthesis. These results provide a mechanistic explanation for the changes in RT activity observed during initiation and show how the dynamics of a ribonucleoprotein complex can regulate enzymatic activity.
ISWI family chromatin remodelers are another family of motor enzymes regulated by nucleic acid structures. These enzymes are involved in creating evenly spaced nucleosome arrays, and this nucleosome spacing activity arises from the regulation of the enzymes’ catalytic activity by the amount of linker DNA present on the nucleosome. We use smFRET and other biochemical assays to monitor intermediates of the remodeling reaction and examine various remodeler mutants in order to elucidate the mechanism of this regulation. These experiments led to the discovery of an allosteric mechanism by which one subunit of the ISWI remodeling complex communicates the presence of linker DNA to the the catalytic subunit by modulating the availability of the histone H4 tail. These results provide a mechanistic explanation for the nucleosome spacing activity of the ISWI chromatin remodelers.
Like the ISWI chromatin remodelers, the SWI/SNF family chromatin remodelers can also reposition nucleosomes, but they may do so by a different mechanism. To investigate the mechanism by which these remodelers move DNA around the nucleosome, we used smFRET to monitor the structural dynamics of nucleosomes during remodeling by the SWI/SNF enzymes. Our results are consistent with movement of the DNA along its canonical path without substantial lifting of DNA off the edges of the nucleosome or displacement of the H2A-H2B dimer. We observe DNA translocation in 1-2 bp increments at both edges of the nucleosome, which suggests that the motion of DNA at the edges of the nucleosome is driven directly by the action of the ATPase near the dyad of the nucleosome.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:23845426
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