RNA-RNA interactions involved in recognition associated with ribozyme catalysis are essential for ribozyme function. Research described in this dissertation focuses on RNA-RNA interactions at, and in the vicinity of two splice sites of two splicing systems: the U2-dependent spliceosome and a yeast mitochondrial group II intron ai5gamma;. In the U2-dependent spliceosomes, an RNA complex is formed by the U2 and U6 snRNAs, creating a network of indispensable helices, which are believed to be the active components of the spliceosome. Helix III has been shown to be essential in mammals. The goal of our study was to analyze structural evidence for formation of U2-U6 Helix III in an in vitro protein-free system, including the possibility of interaction with the intron strand. The questions addressed were: (a) which pairing of the three available strands dominates; (b) alternatively, if the three strands form a complex, is there an RNA triple helix in solution? NMR studies of the three strand complex representing the U2-intron-U6 pairing showed formation of the U2-intron duplex and the U2-U6 duplex, but no interaction of the U6 snRNA with the U2-intron duplex in the region of the putative Helix III. NMR studies of the extended Helix III samples corroborate this finding and confirm that the U6 snRNA immediately upstream of the ACAGAGA sequence does not interact with the U2-intron duplex in the region of the proposed Helix III.
Group II introns, large ribozymes and mobile genetic elements found in prokaryotes and eukaryotic organelles, share common structural and catalytic features with the spliceosome in eukaryotes. At the functional core of group II introns is the pairing of the EBS1-IBS1 sequences (exon binding sequence one and intron binding sequence one, respectively), which is essential for preserving fidelity of the splice site. The EBS1 guide sequence is a part of an 11-nucleotide loop at the terminus of the ID3 stem loop, which is a subdomain of Domain one (D1), the largest of the group II intron domains. The goal of the latter part of this dissertation was to investigate the structural features of the ID3 stem loop and the ID3-IBS1 complex. We investigated the effects of the ID3 stem loop structure on the EBS1-IBS1 pairing. A question addressed was whether the large 11-nucleotide loop forms a stable structure. We tested whether the EBS1-IBS1 pairing forms in solution and what structural changes the ID3 loop undergoes upon formation of the EBS1-IBS1 pairing. We wanted to determine the effects of ID3 structure on the availability of bases of EBS1 for base pairing and thus for the 5' splice site selection. Solution NMR structure of the ID3 stem loop shows a structured stem and a fairly structured base of the loop, as well as an unstructured or dynamic loop, involving residues of the EBS1 sequence. NMR spectroscopic study of the ID3-IBS1 complex in solution indicates that the unstructured region of the ID3 loop becomes structured upon interaction with the IBS1 sequence, in an apparent induced-fit mechanism, by which both the guide sequence and the target become structured upon interaction. An important observation here is that the double stranded EBS1-IBS1 region ends at the 5' splice site, placing it at the single/double stranded junction, which may play an important role in recognition and/or accessibility of the 5' splice site. The placement of the EBS1 sequence in the specific structural context of the ID3 loop may be an important feature, which aids the recognition of the 5' splice site. We show here that by virtue of being placed within the loop of a certain size, the two potential base pairs downstream of the 5' splice site, which could form in a free duplex, do not form in the context of the loop. These findings are important because we show that positioning of a guide sequence within a loop determines the availability of bases for pairing and controls the extent of base pairing and thus the position of the 5' splice site.
|Advisor:||Greenbaum, Nancy L., Logan, Timothy M.|
|Commitee:||Bruschweiler, Rafael P., Li, Hong, Quine, Jack R.|
|School:||The Florida State University|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- Florida|
|Source:||DAI-B 73/12(E), Dissertation Abstracts International|
|Keywords:||Group II intron, Spliceosomes|
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