Structural Biology of RNA by X-ray Crystallography, Chemical Probing, and Cryo-EM
There are a wide variety of non-coding RNAs that fold into well-defined 3D shapes and play important roles in the cell. Despite the importance of these non-coding RNAs in biology, the field of RNA structural biology is not as well-developed as protein structural biology. The main goal of this dissertation is to elucidate the structures of RNA molecules using several approaches. Other work on nucleic acid-related proteins is also presented.
The first part of the dissertation focuses on the group II intron RNA. A crystal structure of the group II intron in the intermediate lariat-3′ exon state was determined to elucidate the mechanism of the second step of splicing and led to a model of the second step in which several junction nucleotides undergo dynamic rearrangements. These dynamic rearrangements are supported by splicing assays of mutants and SHAPE chemical probing. The SHAPE data also revealed that κ-κ′, a tertiary interaction in a different part of the intron, has dynamics that are necessary for splicing.
Chapter 4 looks at the mechanism of selective fidelity in diversity-generating retroelements, a class of genetic elements that can generate a large amount of sequence variability in a protein. This work shows that selective fidelity was due to the low catalytic efficiency of the reverse transcriptase and depended on certain substituents in the nucleobase template.
The next part of the dissertation explores the use of bacterial nanocompartments as a chaperone for cryo-electron microscopy (cryo-EM) structure determination of RNA . First, a high-resolution cryo-EM structure of a thermostable bacterial nanocompartment is presented, illustrating several of its interesting features. Second, a method to assemble RNA inside a nanocompartment is demonstrated and a cryo-EM dataset of this complex was collected, resulting in a 5 Å reconstruction of the encapsulated RNA.
Chapter 7 explores the structure and mechanism of a DNA phosphorothioation complex. This complex is active in vivo and the recombinantly purified proteins bind to DNA in vitro. A cryo-EM dataset of this complex was collected and resulted in a 5 Å density map.