Ribosomal Protein S19 Gene Research Articles

The Drosophila ribosomal protein S6 gene includes a 3' triplication that arose by unequal crossing-over.

Ribosomal protein S6 (rpS6) is the major phosphoprotein of the small ribosomal subunit of eukaryotes and is phosphorylated in response to treatment with mitogens and other stimuli. We have examined the organization of the rpS6 gene of Drosophila melanogaster. Comparisons of a cDNA with genomic DNA identify a transcription unit including three exons. Two tandem repeats downstream of this transcription unit reiterate divergent copies of the third exon and flanking regions. Comparisons of these three repeats with respect to nucleotide base substitutions and deletions or insertions show clearly that they arose via a duplication and subsequent crossing-over between misaligned copies. Although no direct evidence exists that the downstream exons are transcribed, the maintenance of open reading frames in spite of extensive genetic changes is consistent with a protein-coding function.

A small nucleolar RNA is processed from an intron of the human gene encoding ribosomal protein S3.

A human small nucleolar RNA, identified previously in HeLa cells by anti-fibrillarin autoantibody precipitation and termed RNA X, has been characterized. It comprises two uridine-rich variants (148 and 146 nucleotides), which we refer to as snRNA U15A and U15B. Secondary structure models predict for both variants a U15-specific stem-loop structure, as well as a new structural motif that contains conserved sequences and can also be recognized in the other fibrillarin-associated nucleolar snRNAs, U3, U14, and RNA Y. The single-copy gene for human U15A has been found unexpectedly to reside in intron 1 of the ribosomal protein S3 gene; the U15A sequence appears on the same strand as the S3 mRNA and does not exhibit canonical transcription signals for nuclear RNA polymerases. U15A RNA is processed in vitro from S3 intron 1 transcripts to yield the correct 5' end with a 5'-monophosphate; the in vitro system requires ATP for 3' cleavage, which occurs a few nucleotides downstream of the mature end. The production of a single primary transcript specifying the mRNA for a ribosomal or nucleolar protein and a nucleolar snRNA may constitute a general mechanism for balancing the levels of nucleolar components in vertebrate cells.

CDNA nucleotide sequence and expression of a maize cytoplasmic ribosomal protein S13 gene

The complete amino acid sequence of a cytoplasmic ribosomal protein S13 of maize was deduced from the cDNA isolated from a maize cDNA library. The encoded protein is 151 amino acids long and shows a homology of 73% with the corresponding protein S13 of rat. Southern blots analysis shows that the maize protein S13 is encoded by a small multigene family conserved in plant species closely related to maize. The S13 RNAs accumulate preferentially in proliferating tissues and cells and their transcription occurs in parallel to the DNA synthesis.

Characterization of the S7 ribosomal protein gene in wheat mitochondria

By screening a wheat mitoplast cDNA bank, we have identified an open reading frame of 444 bp that has a derived amino acid sequence homologous to bacterial-type S7 ribosomal proteins. This gene, designated rps7, is located upstream of one of two 26S rRNA gene copies in the wheat mitochondrial genome and is expressed as an abundant mRNA of approximately 0.7 kb. Its 5' terminus maps to the end of an 80 bp element that is closely related to sequences preceding the wheat coxII, orf25 and atp6 genes. Southern hybridization analysis indicates that rps7-homologous sequences are present in the mitochondria of rice and pea, but not soybean.

The human ribosomal protein S6 gene: isolation, primary structure and location in chromosome 9

Using PCR cloning we isolated the first intron of the human ribosomal protein S6 gene ( hRPS6). By screening the human HeLa cell cDNA library in λZAPII vector (Stratagene, La Jolla, CA), we identified and sequenced a partially spliced pre mRNA copy of hRPS6. The complete hRPS6 gene was isolated from a λDASH library with an intron-specific probe. The gene and flanking regions were sequenced, and the mRNA 5′ end was mapped by primer extension experiments. The hRPS6 gene has 6 exons and 5 introns and is 3.6 kb long. Using intron-specific primers in PCR and a panel of human-hamster cell lines we localized the hRPS6 gene in human chromosome 9.

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacterium Thermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). The rpsG, fus, and tuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in the str operon of Escherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrograms, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies between Thermotoga and the other bacterial lineages.

A mixed group II/group III twintron in theEuglena gracilischloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts.

Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Structure of the DNA distal to the gene for ribosomal protein S20 inEscherichia coliK12: presence of a strong terminator and an IS1 element

The sequence of nucleotides extending over 2.3 kb distal to the gene for ribosomal protein S20 of E. coli has been determined. Included in the sequence is an efficient rho-independent terminator 50 b.p. distal to the coding sequence for S20, a complete copy of IS1 which lacks, however, flanking direct repeats, and finally, an open reading frame capable of encoding a 28 kDa polypeptide of unknown function. Several lines of evidence suggest that the IS1 sequence described here must represent one of the copies resident in the bacterial chromosome rather than a newly transposed copy. Northern blotting experiments show that the gene for S20 is functionally monocistronic under all conditions tested in several genetic backgrounds. Thus it seems unlikely that the distal copy of IS1 plays any role in the termination or stability of mRNA transcribed from the gene for S20.

Tandem promoters in the gene for ribosomal protein S20.

Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G. A. (1981) J. Biol. Chem. 256, 8177-8182). We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro. In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts. The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate. In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence. A minor start occurs at residue 142 and uses CTP as the initiating nucleotide. In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively. In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder. Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation. Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.

Expression of the gene for ribosomal protein S20: effects of gene dosage.

We have exploited the properties of three different plasmids which carry the gene for Escherichia coli ribosomal protein S20 (rpsT) to test the effects of gene dosage on the expression of rpsT. Over a range of total copies of rpsT of 1 to 58 per haploid genome equivalent, the rate of incorporation of uridine during a 30-s pulse into RNA annealing to either of two specific probes for S20 mRNA increased essentially in proportion to copy number. In contrast, the rate of synthesis of S20 protein increased no more than 2.1-fold at the highest copy number. We conclude, in contrast to an earlier report (D. Geyl, and A. Böck, Mol. Gen. Genet. 154:327-334, 1977), that the synthesis of S20 is regulated at a posttranscriptional step. We propose that S20 itself is the regulatory agent and that binding of S20 to its own mRNA in regions homologous in structure with 16S rRNA can account for our results.

Nucleotide sequence of the gene for ribosomal protein S20 and its flanking regions.

The sequence of almost 700 nucleotides encompassing the gene for ribosomal protein S20 of Escherichia coli has been determined using the chemical technique of Maxam and Gilbert (Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-564). Comparison of this sequence with that of known bacterial promoters reveals two promoter-like sequences whose putative initiation sites for transcription lie 132 ("site 1") and 42 ("site 2") base pairs to the 5' side of the coding sequence. Partial digestion experiments demonstrate that RNA polymerase is capable of binding to and protecting both of these sites from DNase 1 digestion, consistent with their functioning as promoters. Interestingly, site 1 is more compact that any previously described bacterial promoter. A second feature of the sequence is the presence of UUG as the translational initiation codon. Finally, the nucleotide sequence supports the hypothesis (Jue, R. A., Woodbury, N. W., and Doolittle, R. F. (1980) J. Mol. Evol. 15, 129-148) that S20 is composed of three tandemly repeated domains.

The physical localization of the gene for ribosomal protein S20

As a prerequisite to the examination of the structure and properties of the promoter for ribosomal protein S20 of Escherichia coli, I have determined the cleavage sites in λdapB2c I857S7 DNA for four restriction endonuucleases. Subsequently, purified fragments obtained after digestion of this DNA with BamI or HindIII have served as templates for coupled transcription and translation. This has permitted the localization of the structural gene for S20 within a 1000 base pair segment of λdapB2c I857S7 DNA. Cleavage of this DNA with HindIII partially inactivates the expression of S20 in vitro, implying that one HindIII site lies in or near a region essential for the expression of S20.

Synthesis in vitro of ribosomal protein S20 and its precursor

I have purified and characterized two products synthesized in vitro in a system for coupled transcription and translation programmed by DNA from a transducing bacteriophage carrying the gene for ribosomal protein S20. One of these polypeptides appears to be identical with authentic S20 by several criteria, including its electrophoretic and chromatographic mobilities, and its ability to bind to 16S RNA. The second polypeptide is less basic than S20, but exhibits all the structural and functional properties of a precursor to S20, including the presence of an additional methionine residue, apparently as N-formylmethionine. Moreover, it is converted, albeit slowly, to S20 in cell-free extracts. The persistence of the precursor form of S40 may be functionally significant as well.

Isolation of transducing phage carrying rps T, the structural gene for ribosomal protein S20.

Lambda transducing phages carrying segments of the Escherichia coli chromosome in the dapB region have been isolated in their in vivo gene products analyzed by two-dimensional gel electrophoresis. One of these phages, lambdaddapB-2, carries the structural genes for ribosomal protein S20 (rps T) and isoleucyltransfer RNA synthetase (ileS.) The most likely gene order is thr-rpsT-ileS-dapB-pyrA.

Identity of a gene responsible for suppression of aminoacyl-tRNA synthetase mutations with rpsT, the structural gene for ribosomal protein S20.

Sepcialized transducing lines of phage lambda carrying segments between thr and car from the E. coli chromosome have been isolated. With help of these phages it has been shown that the gene sups20 (Böck et al., 1974) corresponds to rpsT, the structural gene for ribosomal protein S20.