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At least 829 records · Page 46Linked to original sources
PubMed1998

Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2.

The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipase A2 (cPLA2-C2) mediates an initial step in the production of lipid mediators of inflammation: the Ca2+-dependent translocation of the enzyme to intracellular membranes with subsequent liberation of arachidonic acid. The high resolution solution structure of this Ca2+-dependent, lipid-binding domain (CaLB) has been determined using heteronuclear three-dimensional NMR spectroscopy. Secondary structure analysis, derived from several sets of spectroscopic data, shows that the domain is composed of eight antiparallel beta-strands with six interconnecting loops that fits the "type II" topology for C2 domains. Using a total of 2370 distance and torsional restraints, the structure was found to be a beta-sandwich in the "Greek key" motif. The solution structure of cPLA2-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholipase-C-delta and phylogenetic analysis clarifies the structural role of highly conserved residues. Calorimetric studies further demonstrate that cPLA2-C2 binds two Ca2+ with observed Kds of approximately 2 microM in an entropically assisted process. Moreover, regions on cPLA2-C2 interacting with membranes were identified by 15N-HSQC-spectroscopy of cPLA2-C2 in the presence of low molecular weight lipid micelles. An extended binding site was identified that binds the phosphocholine headgroup in a Ca2+-dependent manner and also interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of association of cPLA2 with its membrane substrate.

Amino Acid Sequence↗
PubMed2004

Ancient polyploidization predating divergence of the cereals, and its consequences for comparative genomics.

Integration of structural genomic data from a largely assembled rice genome sequence, with phylogenetic analysis of sequence samples for many other taxa, suggests that a polyploidization event occurred approximately 70 million years ago, before the divergence of the major cereals from one another but after the divergence of the Poales from the Liliales and Zingiberales. Ancient polyploidization and subsequent "diploidization" (loss) of many duplicated gene copies has thus shaped the genomes of all Poaceae cereal, forage, and biomass crops. The Poaceae appear to have evolved as separate lineages for approximately 50 million years, or two-thirds of the time since the duplication event. Chromosomes that are predicted to be homoeologs resulting from this ancient duplication event account for a disproportionate share of incongruent loci found by comparison of the rice sequence to a detailed sorghum sequence-tagged site-based genetic map. Differential gene loss during diploidization may have contributed many of these incongruities. Such predicted homoeologs also account for a disproportionate share of duplicated sorghum loci, further supporting the hypothesis that the polyploidization event was common to sorghum and rice. Comparative gene orders along paleo-homoeologous chromosomal segments provide a means to make phylogenetic inferences about chromosome structural rearrangements that differentiate among the grasses. Superimposition of the timing of major duplication events on taxonomic relationships leads to improved understanding of comparative gene orders, enhancing the value of data from botanical models for crop improvement and for further exploration of genomic biodiversity. Additional ancient duplication events probably remain to be discovered in other angiosperm lineages.

Edible Grain↗
PubMed2004

Picornavirus IRES: structure function relationship.

Picornavirus infections have been a challenging problem in human health. Genome organisation of picornavirus is unique in having a long, heavily-structured, multifunctional 5'untranslated region, preceding a single open reading frame from which all viral proteins are produced. Within the 5'leader, an internal region termed ribosome entry site (IRES) regulates viral protein synthesis in a 5'-independent manner. The IRES element itself is a distinctive feature of the picornavirus mRNAs, allowing efficient viral protein synthesis in infected cells in spite of a severe modification of translation initiation factors induced by viral proteases that lead to a fast inhibition of cellular protein synthesis. Picornavirus IRES elements are strongly structured, bearing several motifs, phylogenetically conserved, which are essential for IRES activity. Together with RNA structure, RNA-binding proteins play an essential role in the activity of the IRES element, having a profound effect on viral pathogenesis. Recent data on the involvement of these conserved motifs in RNA structure and protein recognition is discussed in detail. Understanding the interplay between these two components of IRES function is crucial to develop viral strategies aimed to use the viral RNA as the target of antiviral approaches.

5' Untranslated Regions↗
PubMed1997

Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60.

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5' and 3' untranslated transcribed sequences and lack a 3' poly[A] tail. The COI gene does not require uridine insertion/ deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [alpha-32P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3' end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences.

Animals↗
PubMedDate not supplied

[Current concepts regarding the cerebellum and cognition].

OBJECTIVE: To review the main cognitive processes regulated by the cerebellum and the anatomical circuits involved in their clinical correlation. DEVELOPMENT: The cerebellum is generally regarded as a regulator of motor function with a key role in movement coordination. Clinical evidence of the relation of the cerebellum to neural functions typically considered as cortical, is supplied by several neuropsychological alterations detected in both degenerative disorders and acute insult to the region such as vascular event and surgery. More anatomical circuits between the cerebellum and several cortical areas and limbic system, with the ventral pons region as main afference and efference relay of all these pathways. Cerebellar structures that are phylogenetically older such as the floculonodullar lobe, vermix, fastigial nuclei and globus nuclei have a tight relationship to emotional control and autonomic manifestations. More complex circuits are founded in the regulation of learning, motor planning and language. Functional imaging studies have helped to confirm the relationship between the cerebellum and memory processes, finding a selective activation of lateral regions during to cerebellar damage, such a frontal like syndromes, memory deficits and aphasia and even though dysmetria with incoordination between mental process velocity and its motor execution. CONCLUSIONS: All the data from clinical and functional studies indicate that the cerebellum has a central co-ordinating function not only of movement but also regulating thought. The cerebellum should, therefore, be reconsidered as a complex neurone system at much the same level as the more advanced cortical structures.

Cerebellum↗
PubMed2003

The Plasmodium falciparum family of Rab GTPases.

Rab GTPases are key regulators of vesicular traffic in eukaryotic cells. Here we sought a global characterization and description of the Plasmodium falciparum family of Rab GTPases. We used a combination of bioinformatic analyses, experimental testing of predictions, structure modelling and phylogenetics. These analyses led to the identification of seven new parasite Rabs. Accordingly we estimate that the P. falciparum family is made up of 11 genes. We show that ten members of this family are transcribed in infected erythrocytes. Concerning the various members of the family, a series of specific as well as global conclusions can be drawn. Rabs predicted to be compartment-specific show different subcellular distributions. This is demonstrated for PfRab1A and PfRab11A, with the generation of specific antisera. The sequence analyses reveal several peculiarities, with possible functional implications. One of the transcribed genes, Pfrab5b, does not encode a classical C-terminus, suggestive of a novel regulatory role for this GTPase. Another, Pfrab5a, previously identified as a rab gene located on chromosome 2, possesses a 30-amino-acid insertion in its GTP-binding domain. Structural considerations suggest that this insertion could represent a novel interaction interface. We used conserved RabF and RabSF motifs to discriminate between specific parasite Rabs, and followed their predicted change in position on the structure of PfRab6, as GTP is hydrolysed to GDP. This allowed us to propose their involvement in potential interaction surfaces, that we extended to human Rab6 and the motifs known to mediate Rabkinesine-6 binding. Finally, we compared the P. falciparum Rab family to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe and found that parasite Rabs segregate into possible functional clads. Such grouping into clads may give clues to parasite Rab function, and may shed light on P. falciparum secretory/endocytic pathways.

Amino Acid Sequence↗
PubMed2003

SulP, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in Chlamydomonas reinhardtii.

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.

Amino Acid Sequence↗
PubMed2001

Functional and structural similarity of V gamma 9V delta 2 T cells in humans and Aotus monkeys, a primate infection model for Plasmodium falciparum malaria.

Gammadelta T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human gammadelta T cells carrying the Vgamma9Vdelta2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vgamma9Vdelta2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vgamma9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human gammadelta T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vgamma9Vdelta2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.

Amino Acid Sequence↗
PubMed2000

Phylogenetic analysis of molluscan mitochondrial LSU rDNA sequences and secondary structures.

Mollusks are an extraordinarily diverse group of animals with an estimated 200,000 species, second only to the phylum Arthropoda. We conducted a comparative analysis of complete mitochondrial ribosomal large subunit sequences (LSU) of a chiton, two bivalves, six gastropods, and a cephalopod. In addition, we determined secondary structure models for each of them. Comparative analyses of nucleotide variation revealed substantial length variation among the taxa, with stylommatophoran gastropods possessing the shortest lengths. Phylogenetic analyses of the nucleotide sequence data supported the monophyly of Albinaria, Euhadra herklotsi + Cepaea nemoralis, Stylommatophora, Cerithioidea, and when only transversions are included, the Bivalvia. The phylogenetic limits of the mitochondrial LSU rRNA gene within mollusks appear to be up to 400 million years, although this estimate will have to be tested further with additional taxa. Our most novel finding was the discovery of phylogenetic signal in the secondary structure of rRNA of mollusks. The absence of entire stem/loop structures in Domains II, III, and V can be viewed as three shared derived characters uniting the stylommatophoran gastropods. The absence of the aforementioned stem/loop structure explains much of the observed length variation of the mitochondrial LSU rRNA found within mollusks. The distribution of these unique secondary structure characters within mollusks should be examined.

Animals↗
PubMed2002

Peripheral oxygen transport in skeletal muscle of Antarctic and sub-Antarctic notothenioid fish.

Transcellular oxygen flux was modelled mathematically in the aerobic skeletal muscles of perciform fish species living at widely different temperatures (Antarctica, sub-Antarctica and the Mediterranean Sea). Using structural data derived from stereological analysis of electron micrographs, mean fibre P(O(2)) was calculated on the basis of temperature-corrected rates of mitochondrial respiration and oxygen diffusion. The mean muscle fibre diameter (MFD) among Antarctic notothenioids was in the range 17-61 microm and mitochondrial volume density, Vv(mit,f), was 0.27-0.53, but capillary-to-fibre ratio varied only between 1.2 and 1.5. For a mean capillary P(O(2)) of 6 kPa, the model predicted a mean tissue P(O(2)) in the range 0.7-5.8 kPa at the estimated maximum aerobic capacity (M(O(2)max)). The lowest levels of tissue oxygenation were found in the pectoral muscle fibres of the icefish Chaenocephalus aceratus, which lacks the respiratory pigments haemoglobin and myoglobin. Red-blooded notothenioids found in the sub-Antarctic had a similar muscle fine structure to those caught south of the Antarctic Convergence, with an MFD of 20-41 microm and Vv(mit,f) of 0.27-0.33, resulting in an estimated mean P(O(2)) of 4-5 kPa at M(O(2)max). Mean tissue P(O(2)) in the sub-Antarctic icefish Champsocephalus esox, with greater MFD and Vv(mit,f), 56 microm and 0.51, respectively, was calculated to exceed 1 kPa at winter temperatures (4 degrees C), although oxidative metabolism was predicted to be impaired at the summer maximum of 10 degrees C. At the high end of the thermal range, related perciform species from the Mediterranean had a negligible drop in intracellular P(O(2)) across their small-diameter fibres, to a minimum of 5.4 kPa, comparable with that predicted for Trematomus newnesi from the Antarctic (5.6 kPa) with a similar MFD. These data suggest that, within a single phylogenetic group, integrative structural adaptations potentially enable a similar degree of tissue oxygenation over a 20 degrees C range of environmental temperature in the red-blooded notothenioids, and that this is compromised by the lack of respiratory pigments in the icefishes. The mean capillary radius was 1.5 times greater in the two icefish than in the other notothenioids, and the model simulations indicate that the evolution of wide-bore capillaries is essential to maintain tissue oxygenation in the absence of respiratory pigments.

Animals↗
PubMed2001

Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?

Sequencing of the Arabidopsis genome revealed a unique complexity of the plant heat stress transcription factor (Hsf) family. By structural characteristics and phylogenetic comparison, the 21 representatives are assigned to 3 classes and 14 groups. Particularly striking is the finding of a new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs identified from frequently found expressed sequence tags of tomato, potato, barley, and soybean. Evidently, this new type of Hsf is well expressed in different plant tissues. Besides the DNA binding and oligomerization domains (HR-A/B region), we identified other functional modules of Arabidopsis Hsfs by sequence comparison with the well-characterized tomato Hsfs. These are putative motifs for nuclear import and export and transcriptional activation (AHA motifs). There is intriguing flexibility of size and sequence in certain parts of the otherwise strongly conserved N-terminal half of these Hsfs. We have speculated about possible exon-intron borders in this region in the ancient precursor gene of plant Hsfs, similar to the exon-intron structure of the present mammalian Hsf-encoding genes.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of a functional type II gonadotropin-releasing hormone receptor with a lengthy carboxy-terminal tail from an ancestral vertebrate, the sea lamprey.

A full-length transcript encoding a functional type II GnRH receptor was cloned from the pituitary of the sea lamprey, Petromyzon marinus. The current study is the first to identify a pituitary GnRH receptor transcript in an agnathan, which is the oldest vertebrate lineage. The cloned receptor retains the conserved structural features and amino acid motifs of other known GnRH receptors and notably includes a C-terminal intracellular tail of approximately 120 amino acids, the longest C-terminal tail of any vertebrate GnRH receptor identified to date. The lamprey GnRH receptor was shown to activate the inositol phosphate (IP) signaling system; stimulation with either lamprey GnRH-I or lamprey GnRH-III led to dose-dependent responses in transiently transfected COS7 cells. Furthermore, analyses of serially truncated lamprey GnRH receptor mutants indicate perturbations of the C-terminal tail disrupts IP accumulation, however, the tailless lamprey GnRH receptor was not only functional but was also capable of stimulating IP levels equal to wild type. Expression of the receptor transcript was demonstrated in the pituitary and testes using RT-PCR, whereas in situ hybridization showed expression and localization of the transcript in the proximal pars distalis of the pituitary. The phylogenetic placement and structural and functional features of this GnRH receptor suggest that it is representative of an ancestral GnRH receptor. In addition to having an important role in lamprey reproductive processes, the extensive C-terminal tail of this lamprey GnRH receptor may have great significance for understanding the evolutionary change of this vital structural feature within the GnRH receptor family.

Amino Acid Sequence↗
PubMed1998

The correlation of protein structure and evolution of a protein-coding gene: phylogenetic inference using cytochrome oxidase III.

Discriminating phylogenetic signal from noise in DNA sequence data is a difficult problem in phylogenetic inference at higher systematic levels. For protein-coding genes, noise at synonymous (silent) positions can be filtered by deleting entire codon positions or types of change at a codon position. This method is not appropriate for replacement sites, because changes at each site within a codon may not be independent. This research presents a method using information from protein structure to evaluate variation in replacement sites. Analysis of the correlation of amino acid variation with protein structure identified rapidly evolving codons in the COIII gene. In a series of phylogenetic analyses attempting to recover a known set of vertebrate relationships, downweighting these labile codons produced the most accurate results. Structural correlates of variable and invariant residues identified in this study can be used to increase the accuracy of models used for phylogenetic inference. Viewing amino acid variation within a phylogenetic framework provided insight into residue changes important in the secondary and tertiary structures of the molecule, changes that were correlated between pairs of neighboring residues or between residues in neighboring helices.

Amino Acid Substitution↗
PubMed2005

Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon.

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5' and 3' ends of viral genome contained hairpin-like structure length of approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2, the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore propose to rename this virus P. monodon densovirus (PmDNV).

Amino Acid Sequence↗
PubMed1996

Magnesium-aided folding of group I ribozymes with a minimal loss of entropy.

The aim of this work is to quantify, rationalize and incorporate the effect of Mg(II) ions on the kinetic barriers of entropic origin associated to RNA folding events in vitro. This study encompasses the scaffolding effect of Mg(II) upon the formation of the pseudoknot structural motif which is believed to be crucial for shaping the catalytic core of group I ribozymes. Our results are contrasted with recent experimental probes on folding kinetics. The first set of objectives is accomplished by determining the participation of Mg(II) in the reduction of the conformational entropy cost during folding. First, the dominant contribution to conformational entropy loss associated to loop closure is obtained. The derivation hinges upon the notion that loop closure entails the formation of an inner and outer solvent domain and backbone phosphate groups orient themselves concurrently towards the best dielectric environment. At this point, the role of Mg(II) ions can be assessed by taking into account that Mg(II) coordinates with two adjacent backbone phosphates in unpaired regions. Thus, for a small loop of even length, the energy decrease due to coordination reduces the conformational entropy loss due to phosphate orientation by one half. In the case of pseudoknot formation, the orientational effect prevents the coplanarity of the loops comprising the pseudoknot and allows us to determine the kinetic barrier associated to its formation in the presence of magnesium. Incorporating these facts to kinetically controlled Monte Carlo simulations, we find that the predicted folding pathway for group I introns leads to the phylogenetically inferred secondary structures and allows us to elucidate the magnesium-dependent rate-limiting step in the formation of the P3-P7 pseudoknotted region of the catalytic core.

Base Sequence↗
PubMed1991

Long-range structure in ribonuclease P RNA.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

Bacillus subtilis↗
PubMed1995

Phragmoplast of the green alga Spirogyra is functionally distinct from the higher plant phragmoplast.

Cytokinesis in the green alga Spirogyra (Zygnemataceae) is characterized by centripetal growth of a septum, which impinges on a persistent, centrifugally expanding telophase spindle, leading to a phragmoplast-like structure of potential phylogenetic significance (Fowke, L. C., and J. D. Pickett-Heaps. 1969. J. Phycol. 5:273-281). Combining fluorescent tagging of the cytoskeleton in situ and video-enhanced differential interference contrast microscopy of live cells, the process of cytokinesis was investigated with emphasis on cytoskeletal reorganization and concomitant redistribution of organelles. Based on a sequence of cytoskeletal arrangements and the effects of cytoskeletal inhibitors thereon, cytokinetic progression could be divided into three functional stages with respect to the contribution of microfilaments (MFs) and microtubules (MTs): (1) Initiation: in early prophase, a cross wall initial was formed independently of MFs and MTs at the presumptive site of wall growth. (2) Septum ingrowth: numerous organelles accumulated at the cross wall initial concomitant with reorganization of the extensive peripheral interphase MF array into a distinct circumferential MF array. This array guided the ingrowing septum until it contacted the expanding interzonal MT array. (3) Cross wall closure: MFs at the growing edge of the septum coaligned with and extended along the interzonal MTs toward the daughter nuclei. Thus, actin-based transportation of small organelles during this third stage occurred, in part, along a scaffold previously deployed in space by MTs. Displacement of the nuclei-associated interzonal MT array by centrifugation and depolymerization of the phragmoplast-like structure showed that the success of cytokinesis at the third stage depends on the interaction of both MF and MT cytoskeletons. Important features of the phragmoplast-like structure in Spirogyra were different from the higher plant phragmoplast: in particular, MFs were responsible for the positioning of organelles at the fusion site, contrary to the proposed role of MTs in the higher plant phragmoplast.

Cell Division↗
PubMed1998

Structural analysis of the P10/11-P12 RNA domain of yeast RNase P RNA and its interaction with magnesium.

The P10/11-P12 RNA domain of yeast RNase P contains several highly conserved nucleotides within a conserved secondary structure. This RNA domain is essential for enzyme function in vivo, where it has a demonstrated role in divalent cation utilization. To better understand the function of this domain, its structure and alterations in response to magnesium have been investigated in vitro. A secondary structure model of the P10/11-P12 RNA domain had been previously developed by phylogenetic analysis. Computer modeling and energy minimization were applied to the Saccharomyces cerevisiae P10/11-P12 domain to explore alternatives and additional interactions not predicted by the phylogenetic consensus. The working secondary structure models were challenged with data obtained from 1H NMR and in vitro chemical and enzymatic probing experiments. The solution structure of the isolated domain was found to conform to the phylogenetic prediction within the context of the holoenzyme. Structure probing data also discriminated among additional base contacts predicted by energy minimization. The withdrawal of magnesium does not appear to cause gross refolding or rearrangement of the RNA domain structure. Instead, subtle changes occur in the solution accessibility of specific nucleotide positions. Most of the conserved nucleotides reported to be involved in magnesium utilization in vivo also display magnesium-dependent changes in vitro.

Base Sequence↗