In eukaryotes and archaea 5-25% of transfer RNA (tRNA) precursors contain intervening sequences, or introns, that interrupt the molecules' functional regions. Because functional tRNA molecules are necessary for protein synthesis, removing these introns is essential to sustain life. tRNA introns are removed in a two-to-three step process mediated by three different proteins. The RNA splicing endonuclease acts first to cleave two phosphodiester bonds at the intron boundaries within the folded precursor RNAs. The endonuclease performs this function upon nuclear tRNA introns and all archaeal introns. It is well-established that in all organisms the endonuclease step in the splicing pathway is completely conserved, with evidence for the conservation of cleavage chemistry being provided by biochemical studies. However, no detailed information was previously available regarding the endonuclease's specific mechanisms. This research addresses two key aspects of the splicing endonuclease mechanism, namely, substrate recognition and catalysis. Chapter 2 explores the structural elements in a phenotypical archaeal splicing endonuclease and its RNA substrate required for recognition and catalysis. These assays explicitly demonstrate the enzyme and substrate elements involved in recognition and binding. They also support previous findings regarding a conserved triad hypothesized to be catalytic and lay the foundation for the more in-depth studies in Chapter 3. Chapter 3 presents a series of kinetics experiments investigating this conserved triad in which kinetic parameters KM and k2 are obtained. The primary substrate recognition elements in the endonuclease are strictly conserved. However, splicing endonucleases in different organisms are found to have different subunit compositions and substrate specificities. No biochemical studies to date have shed light on how this occurs. Chapter 4 presents studies exploring how the enzyme's quaternary structure affects substrate recognition and cleavage. These studies are continued in Chapter 5, where it is demonstrated that enzyme assembly alone can dictate both substrate specificity and activity. Taken in total, the work presented in this Dissertation provides significant insight regarding how the endonuclease precisely recognizes intron-exon junctions and accelerates the cleavage reaction. It also sheds considerable light on how enzyme subunit composition and quaternary structure relate to the mechanism of RNA recognition. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2007. / April 3, 2007. / Splicing Endonuclease, RNA-Protein Interactions, RNA, Structure-Function Studies, tRNA / Includes bibliographical references. / Hong Li, Professor Directing Dissertation; Myra Hurt, Outside Committee Member; Timothy Cross, Committee Member; Timothy Logan, Committee Member; Brian Miller, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_185023 |
| Contributors | Calvin, Kate (authoraut), Li, Hong (professor directing dissertation), Hurt, Myra (outside committee member), Cross, Timothy (committee member), Logan, Timothy (committee member), Miller, Brian (committee member), Program in Molecular Biophysics (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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