ABSTRACT
Yeast ribosomal protein S14 (rpS14) binds to two different RNA molecules: (1). helix 23 of 18S rRNA during its assembly into 40S ribosomal subunits and (2). a stem-loop structure in RPS14B pre-mRNA to repress expression of the RPS14B gene. We used the three-dimensional structure of Thermus thermophilus ribosomal protein S11, a bacterial homologue of rpS14, as a guide to identify conserved, surface-exposed amino acid residues that are likely to contact RNA. Eight residues that met these criteria were mutated to alanine. Most of these mutations affected interaction of rpS14 with either helix 23 or the RPS14B stem-loop RNA or both. Assembly of 40S ribosomal subunits and repression of RPS14B were also affected. S11 contains an extended carboxy-terminal domain rich in basic amino acids, which interacts with rRNA. We systematically evaluated the importance of each of the last ten amino acid residues in the basic, carboxy-terminal tail of yeast rpS14 for binding to RNA, by mutating each to alanine. Mutations in nine of these residues decreased binding of rpS14 to one or both of its RNA ligands. In addition, we examined the importance of four structural motifs in helix 23 of 18S rRNA for binding to rpS14. Mutations that altered either the terminal loop, the G-U base-pair closing the terminal loop, or the internal loop affected binding of rpS14 to helix 23.
Subject(s)
Bacterial Proteins/chemistry , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA, Ribosomal/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Thermus thermophilus/chemistry , Two-Hybrid System TechniquesABSTRACT
Alu elements, the largest family of interspersed repeats, mobilize throughout the genomes of primates by retroposition. Alu are present in humans in an excess of 500 000 copies per haploid genome. Since some of the insertion alleles have not reached fixation, they remain polymorphic and can be used as biallelic DNA marker systems in investigations of human evolution. In this study, six polymorphic Alu insertional (PAI) loci were used as genetic markers. These markers are thought to be selectively neutral. The presence of these six PAIs was determined by a polymerase chain reaction (PCR)-based assay in 1646 individuals from 47 populations from around the world. Examination of the populations by plotting the first and second principal components, shows the expected segregation of populations according to geographical vicinity and established ethnic affinities. Centroid analysis demonstrated that sub-Sahara populations have experienced higher than average gene flow and/or represent larger populations as compared to groups in other parts of the globe and especially to known inbreed populations. This is consistent with greater heterogeneity and diversity expected of source groups. In addition, maximum likelihood (ML) analyses were performed with these 47 populations and a hypothetical ancestral group lacking the insertion in all six loci. Analysis of our data supports the Out of Africa hypothesis. African populations and admixed groups of African descent formed a single monophyletic group with a basal placement on the tree, which grouped closest to the hypothetical ancestor.
