Molecular characterization of soybean ribosomal protein S13 targeted to the nucleus

We isolated the full-length genomic clone of STLI25 encoding RPS13 (ribosomal protein S13) in soybean. Genomic DNA structure of SLTI25 is similar to that of Arabidopsis ribosomal protein S13. RNA expression of SLTI25 was induced by salt, ABA, or wounding stress, but reduced by dehydration stress. To determine the subcellular localization of the gene product fused to GFP, we were able to confirm that SLTI25-GFP was restricted to the nucleus. By domain swapping analysis, it was shown that C-terminal region of SLTI25 with a putative nucleus localization signal was necessary and sufficient for nucleus targeting of the fusion protein in plants. This new findings provide an evidence that SLTI25 is targeted to the nucleus for the ribosome subunit assembly in plants. Published in Russian in Fiziologiya Rastenii, 2009, vol. 56, No. 3, pp. 445–452 This text was submitted by the authors in English. These authors contributed equally to the work.     

Rapid evolution of goat and sheep globin genes following gene duplication

Statistical analyses of DNA sequences of globin genes (beta A, beta C, and gamma) from goat and sheep (including new sequence information for the second intron of sheep beta A and gamma, kindly provided by A. Davis and A. W. Nienhuis) indicate that the rates of nonsynonymous substitution in these genes have been greatly accelerated following the gene duplication separating gamma and the ancestor of beta A and beta C and the gene duplication separating beta A and beta C. In both cases the acceleration was apparently due to relaxation of purifying selection (functional constraints) rather than advantageous mutations because acceleration occurred only in less important parts of the beta globin chain. The rates of nonsynonymous substitution in these genes are estimated to be about 2.3 x 10(-9) per site per year, which is three times higher than that for the divergence between human beta and mouse beta major globin genes. Our analyses further suggest that the rate of synonymous substitution in functional genes and the rate of substitution in pseudogenes are approximately equal and are between 2.8 x 10(-9) and 5.0 x 10(-9) and that the rate of substitution in introns is about 3.0 x 10(-9). The divergence time between beta A and beta C and that between gamma and the beta A-beta C pair are about 12 and 30 million years, respectively. The proportion of transition mutations is estimated to be 64%, two times higher than expected under random mutation but considerably lower than the 96% estimated for animal mitochondrial DNA.      

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Structural evolution of the Drosophila 5S ribosomal genes

We compare the 5S gene structure from nine Drosophila species. New sequence data (5S genes of D. melanogaster, D. mauritiana, D. sechellia, D. yakuba, D. erecta, D. orena, and D. takahashii) and already-published data (5S genes of D. melanogaster, D. simulans, and D. teissieri) are used in these comparisons. We show that four regions within the Drosophila 5S genes display distinct rates of evolution: the coding region (120 bp), the 5-flanking region (54–55 bp), the 3-flanking region (21–22 bp), and the internal spacer (149–206 bp). Intra- and interspecific heterogeneity is due mainly to insertions and deletions of 6–17-bp oligomers. These small rearrangements could be generated by fork slippages during replication and could produce rapid sequence divergence in a limited number of steps. Correspondence to: M. Wegnez     

The primary structure of rat ribosomal protein S13

The covalent structure of the rat 40S ribosomal subunit protein S13 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Rat S13 contains 150 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 17,080. Hybridization of a S13 cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the gene for the protein. The mRNA for the protein is about 620 nucleotides in length. Rat S13 is related to Saccharomyces cerevisiae YS15 and to Halobacterium marismortui S11. The protein contains a possible internal duplication of 12 residues.     

Human ribosomal protein S13: cloning, expression, refolding, and structural stability

The cDNA of human ribosomal protein S13 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in Escherichia coli cells. Protein accumulated in the form of inclusion bodies was isolated, purified, and refolded by dialysis. The recombinant protein was immunologically reactive, interacting with antiserum against native rpS13. The secondary structure content of the refolded protein was analyzed by means of CD spectroscopy. It was found that 43+/-5% of amino acids sequence of the protein form alpha-helices and 11+/-3% are placed in beta-strands that coincides with theoretical predictions. The beta-strands seem to be located in the extension regions of the rpS13 and do not have homologuous regions in the structure of rpS15 from Thermus thermophilus, which is a prokaryotic homolog of rpS13. The protein structure is stable at a pH range from 4.0 to 8.0 and at low concentrations of urea (up to 3 M).     

Molecular cloning of ribosomal protein genes from Mycoplasma capricolum

Summary A BglII-fragment from the Mycoplasma, capricolum DNA cloned into pBR322 has been found to contain a cluster of ribosomal protein genes. The recombinant plasmid, pMCB1088, includes a 9 kilobase-pair insert that codes for at least eight ribosomal proteins of M. capricolum. The protein genes are expressed in Escherichia coli cells.     

Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch

Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.     

The yeast ribosomal protein S7 and its genes.

Ribosomal protein S7 of Saccharomyces cerevisiae is encoded by two genes RPS7A and RPS7B. The sequence of each copy was determined; their coding regions differ in only 14 nucleotides, none of which leads to changes in the amino acid sequence. The predicted protein consists of 261 amino acids, making it the largest protein of the 40 S ribosomal subunit. It is highly basic near the NH2 terminus, as are most ribosomal proteins. Protein S7 is homologous to both human and rat ribosomal protein S4. RPS7A and RPS7B contain introns of 257 and 269 nucleotides, respectively, located 11 nucleotides beyond the initiator AUG. The splicing of the introns is efficient. Either RPS7A or RPS7B will support growth. However, deletion of both genes is lethal. RPS7A maps distal to CDC11 on chromosome X, and RPS7B maps distal to CUP1 on chromosome VIII.     

Human ribosomal protein S13 inhibits splicing of the own pre-mRNA

Recombinant human ribosomal protein S13 (rpS 13) is shown to bind specifically a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of that fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S16, to this pre-mRNA fragment indicated that the binding of rpS 13 was specific. Besides, poly(AU) and adenovirus pre-mRNA fragment affected poorly the binding of rpS 13 to S13 pre-mRNA, providing another evidence that the interaction was specific. RpS 13 specifically inhibited the pre-mRNA splicing whereas recombinant rpS10 and rpS16 did not affect excision of intron from S13 pre-mRNA fragment in contrast to rpS 13. Those positions in S13 pre-mRNA that were protected by rpS13 protein against cleavage by RNases T1, T2 and V1 were found to be located closely to the 5' and 3' splice sites in the pre-mRNA. Intron 1 in S13 pre-mRNA is more highly conserved within mammals than the other introns in S13 pre-mRNA, which supports the possibility of an important role for intron 1 in the regulation of expression of rpS13 gene in mammals.