Structure of the large ribosomal subunit from human mitochondria.

Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

Evaluation of a novel 16S rRNA/tRNAVal mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea.

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)(Val) genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA(Val) genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA(Val) marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA(Val) gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA(Val) region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA(Val) marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.

A polymerase chain reaction-restriction fragment length polymorphism method based on the analysis of a 16S rRNA/tRNA(Val) mitochondrial region for species identification of commercial penaeid shrimps (Crustacea: Decapoda: Penaeoidea) of food interest.

A simple PCR-RFLP method has been developed for the identification of 19 penaeid shrimp species of food interest belonging to the superfamily Penaeoidea. Preliminary amplification, sequencing and alignment of a 960 bp fragment of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial region allowed the design of 16Scru4/16Scru4 primers, constructed on well-conserved mitochondrial sequences of the penaeid shrimp species considered. Such primers afforded the amplification of an internal 515-535 bp region of the 16S rRNA/tRNA(Val) genes that, when subjected to cleavage with AluI, TaqI and HinfI, provided species-specific restriction patterns. Moreover, the proposed method also allowed the definition of different intraspecific restriction types between different populations of Litopenaeus vannamei, Farfantepenaeus notialis, Fenneropenaeus merguiensis, Metapenaeus sp., Melicertus latisulcatus and Pleoticus muelleri of different origins. The method described here was also successfully applied for the identification of penaeid shrimps in complex processed precooked foods, where this type of shellfish is used as an added-value food ingredient. Sequencing analysis provided new information about the genetic relationships among shrimps not only at the levels of species and genus, but also among different populations at intraspecific level. The 16S rRNA/tRNA(Val) fragment considered in this study seems to be accurate for shrimp species identification in raw and processed foodstuffs and for phylogenetic analysis among penaeid shrimp species.

The early history of modern birds inferred from DNA sequences of nuclear and mitochondrial ribosomal genes.

The traditional view of avian evolution places ratites and tinamous at the base of the phylogenetic tree of modern birds (Neornithes). In contrast, most recent molecular studies suggest that neognathous perching birds (Passeriformes) compose the oldest lineage of modern birds. Here, we report significant molecular support for the traditional view of neognath monophyly based on sequence analyses of nuclear and mitochondrial DNA (4.4 kb) from every modern avian order. Phylogenetic analyses further show that the ducks and gallinaceous birds are each other's closest relatives and together form the basal lineage of neognathous birds. To investigate why other molecular studies sampling fewer orders have reached different conclusions regarding neognath monophyly, we performed jackknife analyses on our mitochondrial data. Those analyses indicated taxon-sampling effects when basal galloanserine birds were included in combination with sparse taxon sampling. Our phylogenetic results suggest that the earliest neornithines were heavy-bodied, ground-dwelling, nonmarine birds. This inference, coupled with a fossil bias toward marine environments, provides a possible explanation for the large gap in the early fossil record of birds.

Molecular cloning of GAF2, a Schizosaccharomyces pombe GATA factor, which has two zinc-finger sequences.

By low stringency screening of a lambda-Shizosaccharomyces pombe genomic library, we have cloned a GATA factor homologous gene, gaf2+, within a 3.1-kb EcoRI fragment. The gaf2 ORF predicts a protein of M(r) 61 kDa consisting of intronless 564 amino acids corresponding to 1,692 bp. Gaf2 has two zinc-fingers as Urbs1 of Ustilago maydis, whereas most of fungal GATA factors have only one zinc-finger. The separation between two zinc-fingers of Gaf2 is rather long. In addition to gaf2, the sequence analysis revealed a Val-tRNA gene in the 3'-flanking region of gaf2. Northern blot analysis indicated that the gaf2 gene is transcribed constitutively irrespective of the nitrogen source in a medium.

Valyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Val)-C-C-A and tRNA(Val)-C-C-A(3'NH2).

For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2-amino-isobutyric acid by valyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNA(Val)-C-C-A. The lowest values were found for Trp, Ser, Cys, Lys, Met and Thr (D = 90-870), showing that valine is 90-870 times more frequently attached to tRNA(Val)-C-C-A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1,100 and 6200. Generally, valyl-tRNA synthetase is considerably less specific than isoleucyl-tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids. In aminoacylation of tRNA(Val)-C-C-A(3'NH2) discrimination factors D1 are in the range of 40-1260. From D1 values and AMP formation stoichiometry, pretransfer proof-reading factors pi 1 were determined: post-transfer proof-reading factors II 2 were determined from D values and AMP formation stoichiometry in acylation of tRNA(Val)-C-C-A. II 1 values (7-168) show that pretransfer proof-reading is the main correction step, post-transfer proof-reading (II 2 approximately 1-7) is less effective and in some cases negligible. Initial discrimination factors were calculated from discrimination and proof-reading factors according to a two-step binding process. These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical 'stopper' model of the amino-acid-binding site is discussed.

Genes, variant genes and pseudogenes of the human tRNA(Val) gene family. Expression and pre-tRNA maturation in vitro.

Nine different members of the human tRNA(Val) gene family have been cloned and characterized. Only four of the genes code for one of the known tRNA(Val) isoacceptors. The remaining five genes carry mutations, which in two cases even affect the normal three-dimensional tRNA structure. Each of the genes is transcribed by polymerase III in a HeLa cell nuclear extract, but their transcription efficiencies differ by up to an order of magnitude. Conserved sequences immediately flanking the structural genes that could serve as extragenic control elements were not detected. However, short sequences in the 5' flanking region of two genes show striking similarity with sequences upstream from two Drosophila melanogaster tRNA(Val) genes. Each of the human tRNA(Val) genes has multiple, i.e. two to four, transcription initiation sites. In most cases, transcription termination is caused by oligo(T) sequences downstream from the structural genes. However, the signal sequences ATCTT and CTTCTT also serve as effective polymerase III transcription terminators. The precursors derived from the four tRNA(Val) genes coding for known isoacceptors and those derived from two mutant genes are processed first at their 3' and subsequently at their 5' ends to yield mature tRNAs. The precursor derived from a third mutant gene is incompletely maturated at its 3' end, presumably as a consequence of base-pairing between 5' and 3' flanking sequences. Finally, precursors encoded by the genes that carry mutations affecting the tRNA tertiary structure are completely resistant to 5' and 3' processing.

An efficiently transcribed human tRNA(Val) gene variant produces a stable pre-tRNA: repair of the processing defect by in vitro mutations.

A tRNA(CACVal) gene variant, pHtV4, was cloned from human placenta genomic DNA. This gene differs from a closely related, functional tRNA(CACVal) gene by four base exchanges: T residues in place of C25, C62, and C66 create G:U pairs, and an A instead of G65 creates an A:C mismatch in the corresponding RNA transcript. The tRNA(Val) gene variant in pHtV4 is efficiently transcribed in HeLa cell nuclear extracts; however, the resulting pre-tRNA is processing-deficient, i.e., neither its 5'- nor its 3'-flanking sequences are removed to generate mature tRNA. Reversion of all four point mutations in pHtV4 by oligonucleotide-directed mutagenesis yielded a functional tRNA(CACVal) gene within the flanks of pHtV4, the pre-tRNA of which was processed to mature tRNA. Construction of a chimeric tRNA(Val) gene and site-directed mutagenesis of the tRNA(Val) gene in pHtV4, respectively, followed by transcription and processing studies showed that each of the four mutations contributes to the processing defect of the pHtV4-derived pre-tRNA. Moreover, this revealed that G:U pairs, which are common in all tRNAs, can impair pre-tRNA processing and therefore do not occur in certain positions in eukaryotic tRNAs.

Valylation of tRNA-like transcripts from cloned cDNA of turnip yellow mosaic virus RNA demonstrate that the L-shaped region at the 3' end of the viral RNA is not sufficient for optimal aminoacylation.

Clones containing different lengths of cDNA corresponding to the 3' region of turnip yellow mosaic virus RNA were constructed and transcribed in vitro into the corresponding RNAs. Each transcript contained the L-shaped tRNA domain (N = 82) plus (i) in the case of 3 upstream sequences up to N = 93, 109 and 258; and (ii) in all cases an additional 6 nucleotide-stretch at the 5' end derived from the T7 promoter. The valylation of these molecules, as well as that of a fragment (N = 159) purified from viral RNA, was studied. Although all transcripts could be valylated by wheat germ valyl-tRNA synthetase, the 3 shorter fragments showed incomplete charging and slower rates, due mainly to lower Vmax values. Thus, although the tRNA-like L-shaped structure is the functional core permitting amino-acylation, upstream nucleotides between positions 82 and 159 play an important role in allowing the highest rates and levels of valylation. Structural arguments supporting this view are discussed.