The experiments were focussed on three protein-nucleic acid interactions in the Xenopus oocyte: TFIIIA-5S RNA, TFIIIA-5S DNA and ribosomal protein L5-5S RNA. The binding affinities and contact sites of the proteins to the nucleic acids were studied.
For studying the TFIIIA-5S RNA interaction, block mutations were constructed in helical stems II, III, IV and V of Xenopus laevis oocyte 5S RNA. The affinities of these mutants for binding to transcription factor IIIA were determined using a nitrocellulose filter binding assay. Mutations in stems III and IV had little or no effect on the binding affinity of TFIIIA for 5S RNA. However, single mutants in stems II and V (positions 16-21, 57-62, 71-72, and 103-104) which disrupt the double helix, reduce the binding of TFIIIA by a factor of two- to three-fold. In contrast, double mutants (16-21/57-62, 71-72/103-104) which restore the helical structure of these stems, but with altered sequences, fully restore the TFIIIA binding affinity. The experiments reported here indicate that the double helical structures of stems II and V, but not the sequences, are required for optimal TFIIIA binding.
The effects on TFIIIA binding affinity of a series of substitution mutations in the Xenopus laevis oocyte 5S RNA gene were quantified. These data indicate that TFIIIA binds specifically to 5S DNA by forming sequence-specific contacts with three discrete sites located within the classical A and C boxes and the intermediate element of the internal control region. Substitution of the nucleotide sequence at any of the three sites significantly reduces TFIIIA binding affinity, with a 100-fold reduction observed for substitutions in the box C subregion. These results are consistent with a direct interaction of TFIIIA with specific base pairs within the major groove of the DNA. In contrast, the TFIIIA binding data for the same mutations expressed in 5S RNA indicates that the protein does not make any strong sequence-specific contacts with the RNA. Although the protein footprinting sites on the 5S DNA and 5S RNA are coincident, nucleotide substitutions in 5S RNA which moderately reduce TFIIIA binding affinity do not correspond at all to the three specific TFIIIA interaction sites within the gene.
For investigating the L5-5S RNA interaction, a cDNA encoding ribosomal protein L5 of Xenopus laevis was subcloned into a T7 expression vector and expressed in Escherichia coli. The resulting soluble fusion protein with a histidine tag at the N-terminus was purified by affinity chromatography to 95% homogeneity. The equilibrium binding of recombinant L5 to Xenopus 5S ribosomal RNA was characterized, and the affinity of the protein for a set of 5S RNA mutants was quantitatively measured using a nitrocellulose filter binding assay. L5 binds to 5S RNA with properties similar to those of the TFIIIA-5S RNA interaction. However, unlike TFIIIA, L5 was insensitive to changes in either the sequence or the secondary structure of the 5S RNA.
The results from these studies indicate that the specific protein-nucleic acid interactions in the biological pathway of 5S RNA use distinct mechanisms. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9610 |
| Date | 05 July 2018 |
| Creators | You, Qimin |
| Contributors | Romaniuk, Paul John |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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