The Further Characterization of Saccharomyces cerevisiae Ribosomal Protein L30 and Its Interaction with the RPL30 Transcript through Mutational Analysis, Circular Dichroism, and Fluorescence Methods
type: CreativeWork
description: The Saccharomyces cerevisiae ribosomal protein L30 is one of a unique group of proteins that bind to their own mRNA in a feedback regulation mechanism. L30 binds to a stem-loop-stem region in the RPL30 pre-mRNA which is called the kink-turn motif. This motif is found in several protein-binding RNAs in the ribosome, as well as in some small ribonucleoprotein particles such as components of the spliceosomal assembly. In order to study the interaction of L30 with its RPL30 kinkturn, several mutations have been made in L30 protein at key positions in the protein/ RNA interface. Phenylalanine 85 has been shown to provide a stabilizing stacking interaction with the purine-rich kink-turn loop in the RPL30 transcript. Using past NMR and current crystallographic structural information about yeast L30 and phylogenetic comparison of L30 proteins in other species, single amino acid mutations at positions 84 and 85 in L30 protein were designed to study the effect of those changes on RNA/protein binding. Amino acids were selected for mutation to establish the necessity of an aromatic and hydrophobic residue in L30 position 85. The phenylalanine in position 85 was mutated to tryptophan, histidine, and isoleucine and the dissociation constant of each mutated L30 was compared to that of the wild type L30 using radioactive filter binding and band shift assays. To further study the position 85 region of L30, single alanine insertions at positions 84 and 86 and single amino acid mutations at position 84 such as proline, histidine and tyrosine were also made and assayed using filter binding and band shift experiments. In order of strongest binding to least, the position 85 mutants were histidine, tryptophan, and isoleucine, with Kd values of 4.3 nM, 10.5 nM, and 58.7 nM, respectively. Both alanine insertions exhibited decreased binding with Kd values for A84 and A86 being 58.2 nM and 170.4 nM, with the insertion at position 86 being the most disruptive. These results suggested that the …
abstract: The Saccharomyces cerevisiae ribosomal protein L30 is one of a unique group of proteins that bind to their own mRNA in a feedback regulation mechanism. L30 binds to a stem-loop-stem region in the RPL30 pre-mRNA which is called the kink-turn motif. This motif is found in several protein-binding RNAs in the ribosome, as well as in some small ribonucleoprotein particles such as components of the spliceosomal assembly. In order to study the interaction of L30 with its RPL30 kinkturn, several mutations have been made in L30 protein at key positions in the protein/ RNA interface. Phenylalanine 85 has been shown to provide a stabilizing stacking interaction with the purine-rich kink-turn loop in the RPL30 transcript. Using past NMR and current crystallographic structural information about yeast L30 and phylogenetic comparison of L30 proteins in other species, single amino acid mutations at positions 84 and 85 in L30 protein were designed to study the effect of those changes on RNA/protein binding. Amino acids were selected for mutation to establish the necessity of an aromatic and hydrophobic residue in L30 position 85. The phenylalanine in position 85 was mutated to tryptophan, histidine, and isoleucine and the dissociation constant of each mutated L30 was compared to that of the wild type L30 using radioactive filter binding and band shift assays. To further study the position 85 region of L30, single alanine insertions at positions 84 and 86 and single amino acid mutations at position 84 such as proline, histidine and tyrosine were also made and assayed using filter binding and band shift experiments. In order of strongest binding to least, the position 85 mutants were histidine, tryptophan, and isoleucine, with Kd values of 4.3 nM, 10.5 nM, and 58.7 nM, respectively. Both alanine insertions exhibited decreased binding with Kd values for A84 and A86 being 58.2 nM and 170.4 nM, with the insertion at position 86 being the most disruptive. These results suggested that the …
author: Selah, Cheryl A.
keywords: Chemistry
date published: 2007-01-01T08:00:00Z
date modified: 2026-07-24T01:23:36Z
provider: Bryn Mawr University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Bryn Mawr University