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PubMed2003

Conserved signature proposed for folding in the lipocalin superfamily.

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model beta-barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol-binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin beta-lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time-scale than the non-conserved regions.

Circular Dichroism↗

PubMed2000

Sequence conservation in kringle IV-type 2 repeats of the LPA gene.

We have studied the homology of repeating kringle IV-type 2 (K IV-type 2) elements of the LPA gene. Two K IV-type 2 genomic polymerase chain reaction (PCR) fragment libraries were constructed, one from an individual with high and one from an individual with low Lp(a) lipoprotein level. Only minor K IV-type 2 repeat length heterogeneity was observed. Sequence analysis data from the cloned K IV-type 2 repeats revealed a high degree of LPA sequence conservation in exons as well as in introns both within and between the two libraries. This sequence conservation of the IV-type 2 kringles is in agreement with our previously reported results of simultaneous 'batch' DNA sequence analyses of all the K IV-type 2 repeats from single individuals. Sequence data from the clones, combined with genomic DNA sequencing, revealed that the K IV-type 2 reading frame of exons 1 and 2 are extended into the conserved flanking introns by 519 base pairs (bp) and 312 bp, respectively. The theoretical coding capacity of the exon 1 extended open reading frame (ORF I) is three times larger (173 amino acids, aa) than the translated exon 1, and that of the extended open reading frame of exon 2 (ORF II) is about twice (104 aa) the length of exon 2. A central portion of the intron separating exons 1 and 2 also exhibited a high degree of sequence conservation, with the exception of a polymorphic CA repeat. Within the 61 K IV repeat clones analysed, 19 different CA repeat patterns with 12-18 CA dinucleotide repeats were observed. A comparison between the 37 clones from the individual with high Lp(a) lipoprotein level and the 24 clones from the individual with low Lp(a) lipoprotein level, revealed that seven of the CA repeat variants were present in both clone libraries. The observed high level of sequence conservation in K IV-type 2 exons and introns matches relevant areas of the plasminogen gene, and our findings fit with recent K IV-type 2 duplications and evolutionary selection pressure theories, although gene conversion events could also explain the findings. DNA sequences within K IV-type 2 appeared to have no influence on Lp(a) lipoprotein level.

Adolescent↗

PubMed1994

A beta-tubulin gene of Naegleria encodes a carboxy-terminal tyrosine. Aromatic amino acids are conserved at carboxy termini.

A gene that directs the programmed synthesis of flagellar beta-tubulin during the rapid differentiation of Naegleria gruberi from amoebae to flagellates has been cloned and sequenced. The intronless gene is one of 8 to 10 similar but non-identical genes that are dispersed in the genome. beta-Tubulin mRNA homologous to this gene family is expressed transiently during differentiation, and has not been detected in amoebae. The encoded beta-tubulin is strongly conserved, with features that closely resemble the beta-tubulins of diverse organisms, especially organisms that, like Naegleria, use tubulin to assemble flagellar axonemes. In most sequenced alpha-tubulins, the encoded carboxy-terminal amino acid is tyrosine, which undergoes post-translational removal and readdition, conserved processes of unknown function. In N. gruberi, unusually, the terminus of alpha-tubulin is encoded as glutamine while that of beta-tubulin is tyrosine. The presence of these divergent termini on subunits of a conserved tubulin provoked us to re-examine aromatic amino acids at the termini of alpha- and beta-tubulins. Although evolution has tinkered extensively with the carboxy-terminal domains of tubulin subunits, we find an unexpected conservation. In every organism or cell type for which both tubulin subunits have been sequenced, except the ciliate Stylonychia lemnae, at least one tubulin subunit of some or all tubulin heterodimers terminates in an aromatic amino acid, either tyrosine or phenylalanine. This remarkable conservation of carboxy-terminal aromatic amino acids suggests that these residues serve some crucial function.

Amino Acid Sequence↗

PubMed2003

DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus.

Nonstructural protein 3 (NS3) of human hepatitis C virus (HCV) is a conserved multi-functional protein essential for replication and translation of viral RNA and polyprotein processing. Early T-cell response against NS3 is capable of restricting viremia. We aimed at characterizing the immunogenicity in gene immunization of the conserved regions of NS3 critical for protein folding and activity. C57BL/6 mice were injected with NS3 gene of Russian HCV 1b isolate 274933RU. Immunization did not exert any overt histological changes and had no long-term effects on the immune status of NS3 gene-recipients. The immune response in NS3 gene-recipients was screened by antibody ELISA, T-cell proliferation test and immune assays for specific cytokine production. T-lymphocytes of NS3 gene-recipients proliferated in response to peptides representing conserved regions of protease and ATPase/helicase. Stimulated T-lymphocytes produced IL-2, and in response to protease-derived peptides, also IFN-gamma. Potent and long-lasting antibody response was raised against conserved NS3 regions including "Greek-key" motif of protease, motifs II, V and polynucleotide-binding domains of ATPase/helicase. Thus, gene immunization effectively targeted conserved regions critical for NS3 protease and helicase function. In type and specificity, immune response of NS3 gene-immunized mice mimicked immunity achieved in the acute self-limiting HCV infection of human and primates and in virus-exposed healthy individuals, indicating promiscuity of NS3 as immunogen.

Adenosine Triphosphatases↗

PubMed2000

Identification and characterization of conserved cis-regulatory elements in the human keratocan gene promoter.

Keratocan, along with lumican and mimecan, represent the keratan sulfate-containing proteoglycans of the vertebrate cornea that play a key role in development and maintenance of corneal transparency. In this study, we cloned 4.1 kb of the human Kera 5'-flanking region and characterized the promoter structure. Using primer extension and ribonuclease protection assay, we identify two major transcriptional start sites in the first exon. Using luciferase reporter gene transfection analysis of 5'-deletion and mutation constructs, we demonstrate positive and negative regulatory elements within a 1.3 kb upstream sequence. Comparison of human and bovine 5'-flanking sequences reveals three highly conserved regions: a 450 bp region in the first exon, a 92 bp promoter proximal conserved regulatory region identified as an enhancer in the natural context, and a 223 bp promoter distal conserved regulatory region identified as a silencer both in the natural context and in a heterologous promoter system. In addition, a conserved CArG-box residing 851 bp upstream of the first transcription start site also can lead to the repression of Kera expression in cultured corneal keratocytes. DNaseI footprinting and electrophoretic mobility shift assay demonstrate that cell type-specific factors bind to regulatory elements located in the conserved regions. Competition experiments indicate that the CTC factor and a protein that binds to the CAGA motif are likely to be among the multiple factors involved in the transcriptional regulation of the human Kera gene.

Animals↗

PubMed1998

Roles of conserved residues in the arginase family.

Arginases and related enzymes metabolize arginine or similar nitrogen-containing compounds to urea or formamide. In the present report a sequence alignment of 31 members of this family was generated. The alignment, together with the crystal structure of rat liver arginase, allowed the assignment of possible functional or structural roles to 32 conserved residues and conservative substitutions. Two of these residues were previously identified as functionally essential by analysis of inherited defects in the type I arginase gene. Nearly half of the conserved residues are either glycines or prolines located at critical bends in the protein structure. Most metal-coordinating residues, including one histidine and four aspartic acid residues, are strictly conserved. Two additional histidines involved in metal-binding and catalysis are conserved in all arginases and in almost all other family members. Two positions with invariant similarities may serve as indirect metal ligands. Evolutionary relationships within this family were also suggested. Vertebrate type I and II arginases appear to have developed independently from an early gene duplication event. A ureohydrolase sequence from Caenorhabditis elegans is more closely related to other arginases than previously appreciated, while unclassified enzymes from Methanococcus jannaschii and Methanothermus fervidus appear more similar to arginase-related enzymes. In addition, enzymes from Arabidopsis thaliana and Synechocystis, previously identified as arginases, more closely resemble arginase-related enzymes than currently known arginases.

Amino Acid Sequence↗

PubMed2003

A significant fraction of conserved noncoding DNA in human and mouse consists of predicted matrix attachment regions.

Noncoding DNA in the human-mouse orthologous intergenic regions contains "islands" of conserved sequences, the functions of which remain largely unknown. We hypothesized that some of these regions might be matrix-scaffold attachment regions, MARs (or S/MARs). MARs comprise one of the few classes of eukaryotic noncoding DNA with an experimentally characterized function, being involved in the attachment of chromatin to the nuclear matrix, chromatin remodeling and transcription regulation. To test our hypothesis, we analyzed the co-occurrence of predicted MARs with highly conserved noncoding DNA regions in human-mouse genomic alignments. We found that 11% of the conserved noncoding DNA consists of predicted MARs. Conversely, more than half of the predicted MARs co-occur with one or more independently identified conserved sequence blocks. An excess of conserved predicted MARs is seen in intergenic regions preceding 5' ends of genes, suggesting that these MARs are primarily involved in transcriptional control.

Animals↗

PubMedDate not supplied

Mutation of highly conserved arginine residues disrupts the structure and function of annexin V.

BACKGROUND: Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). METHODS: To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. RESULTS: Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca(2+)-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 microM Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. CONCLUSIONS: Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges.

Amino Acid Sequence↗

PubMed1998

Prediction of well-conserved HIV-1 ligands using a matrix-based algorithm, EpiMatrix.

This preliminary study was undertaken to identify new human leucocyte antigens (HLA) ligands from human immunodeficiency virus type 1 (HIV-1) which are highly conserved across HIV-1 clades and which may serve to induce cross-reactive cytotoxic T lymphocytes (CTLs). EpiMatrix was used to predict putative ligands from HIV-1 for HLA-A2 and HLA-B27. Twenty-six peptides that were both likely to bind and also highly conserved across HIV-1 strains in the Los Alamos HIV sequence database were selected for binding assays using the T2 stabilization assay. Two peptides that were also highly likely to bind (for A2 and B27, as determined by EpiMatrix) and well conserved across HIV-1 strains, and had previously been described to bind in the published literature, were also selected to serve as positive controls for the assays. Ten new major histocompatibility complex (MHC) ligands were identified among the 26 study peptides. The control peptides bound, as expected. These data confirm that EpiMatrix can be used to screen HIV-1 protein sequences for highly conserved regions that are likely to bind to MHC and may prove to be highly conserved HIV-1 CTL epitopes.

Algorithms↗

PubMed2000

Co-conservation of rRNA tetraloop sequences and helix length suggests involvement of the tetraloops in higher-order interactions.

Terminal loops containing four nucleotides (tetraloops) are common in structural RNAs, and they frequently conform to one of three sequence motifs, GNRA, UNCG, or CUUG. Here we compare available sequences and secondary structures for rRNAs from bacteria, and we show that helices capped by phylogenetically conserved GNRA loops display a strong tendency to be of conserved length. The simplest interpretation of this correlation is that the conserved GNRA loops are involved in higher-order interactions, intramolecular or intermolecular, resulting in a selective pressure for maintaining the lengths of these helices. A small number of conserved UNCG loops were also found to be associated with conserved length helices, consistent with the possibility that this type of tetraloop also takes part in higher-order interactions.

Base Sequence↗

PubMed2003

Economic growth, biodiversity loss and conservation effort.

This paper investigates the relationship between economic growth, biodiversity loss and efforts to conserve biodiversity using a combination of panel and cross section data. If economic growth is a cause of biodiversity loss through habitat transformation and other means, then we would expect an inverse relationship. But if higher levels of income are associated with increasing real demand for biodiversity conservation, then investment to protect remaining diversity should grow and the rate of biodiversity loss should slow with growth. Initially, economic growth and biodiversity loss are examined within the framework of the environmental Kuznets hypothesis. Biodiversity is represented by predicted species richness, generated for tropical terrestrial biodiversity using a species-area relationship. The environmental Kuznets hypothesis is investigated with reference to comparison of fixed and random effects models to allow the relationship to vary for each country. It is concluded that an environmental Kuznets curve between income and rates of loss of habitat and species does not exist in this case. The role of conservation effort in addressing environmental problems is examined through state protection of land and the regulation of trade in endangered species, two important means of biodiversity conservation. This analysis shows that the extent of government environmental policy increases with economic development. We argue that, although the data are problematic, the implications of these models is that conservation effort can only ever result in a partial deceleration of biodiversity decline partly because protected areas serve multiple functions and are not necessarily designated to protect biodiversity. Nevertheless institutional and policy response components of the income biodiversity relationship are important but are not well captured through cross-country regression analysis.

Animals↗

PubMed1999

Conserved amino acids in the ligand-binding and tau(i) domains of the peroxisome proliferator-activated receptor alpha are necessary for heterodimerization with RXR.

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. These ligand-activated transcription factors are implicated in the regulation of lipid metabolism and adipocyte differentiation and in the regulation of anti-inflammatory processes. In order to bind to DNA and activate transcription PPAR requires the formation of heterodimers with the retinoid X receptor (RXR). We have previously reported that replacement of a single leucine by an arginine at position 433 of hPPAR alpha (L433R), located in a highly conserved region of the ninth heptad repeat of a leucine-zipper-like motif in the ligand binding domain, abolished heterodimerization of PPAR with RXR and hence its trans-activating capacity. The aim of our present work was to investigate if other conserved amino acids of the ligand binding domain are important for heterodimerization of PPAR with RXR. We found that conserved leucines, L370 and L391, in a leucine-zipper-like motif of hPPAR alpha, as well as a highly conserved aspartic acid (D304) in the tau(i) domain are necessary for heterodimerization with RXR. In contrast, mutations of non-conserved amino acids within the leucine-zipper-like motif do not affect PPAR:RXR heterodimerization. Surprisingly, we found that some mutants deficient in heterodimerization with RXR (hPPAR alpha-L370R and -L391R) were still functional on specific peroxisome proliferator-activator response elements (PPREs). Both mutants could trans-activate on a PPRE from the P450 cytochrome promoter CYP4A1, whereas only the hPPAR alpha-L391R mutant could trans-activate from the acyl-CoA oxidase PPRE (ACOA) and, when stimulated with the peroxisome proliferator Wy14643, also from the bifunctional enzyme PPRE. We therefore hypothesize either that: (i) these mutants might be able to heterodimerize with a protein other than RXR and the affinity for this novel partner may depend on the nature of the PPRE and to some degree on the choice of the activator, or alternatively; (ii) that additional nuclear proteins might compensate in vivo for the decreased binding of RXR to these mutant PPARs observed in vitro.

Acyl-CoA Oxidase↗

PubMed2003

Transcription control of a gene for Drosophila transcription factor, DREF by DRE and cis-elements conserved between Drosophila melanogaster and virilis.

A DNA replication-related element (DRE)-binding factor (DREF) has been revealed to be an important transcription factor for activating promoters of cell proliferation and differentiation related genes. The amino acid sequences of DREF are conserved in evolutionary separate Drosophila species, Drosophila melanogaster (Dm) and Drosophila virilis (Dv) in three regions. In the present study, evidence was obtained that there are several highly conserved regions in the 5' flanking region between the DmDREF and DvDREF genes. Band mobility shift assays using oligonucleotides corresponding to these conserved regions revealed that specific trans-acting factors can bind to at least three regions -554 to -543 (5'-TTTGTTCTTGCG), -81 to -70 (5'-GCCCACGTGGCT) and +225 to +234 (5'-GCAATCAGTG). Using a transient luciferase expression assay, we demonstrated that the region -554 to -543 functions as a negative regulatory element for DmDREF promoter activity, while the regions -77 to -70 (5'-ACGTGGCT) and +225 to +236 (5'-GCAATCAGTGTT) function as positive regulatory elements. In previous studies, we observed that expression of the homeodomain protein Zerknüllt (Zen) represses PCNA gene transcription, by reducing the DNA binding activity of DREF. Here we show Zen downregulates DREF gene promoter activity through action on the region between +241 and +254 (5'-AGAATACTCAACA). In addition, the DmDREF promoter contains five DREs. Using a double stranded RNA-mediated interference method, we generated evidence that expression of DmDREF could be auto-regulated by DREF through the third DRE located at +211 to +218. In living flies we obtained results consistent with those obtained in vitro and in cultured cells. The study thus indicates that DmDREF is effectively regulated via highly conserved regions between the DmDREF and DvDREF promoters, suggesting the existence of common regulatory factors, and that DmDREF can be positively regulated by itself via the third DRE located in its most highly conserved region.

5' Flanking Region↗

PubMed1997

Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications.

This paper reports the first comprehensive analysis of Displacement loop (D-loop) region sequences from ten different mammalian orders. It represents a systematic evolutionary study at the molecular level on regulatory homologous regions in organisms belonging to a well defined class, mammalia, which radiated about 150 million years ago (Mya). We have aligned and analyzed 26 complete D-loop region sequences available in the literature and the fat dormouse sequence, recently determined in our laboratory. The novelty of our alignment consists of the extensive manual revision of the preliminary output obtained by computer program to optimize sequence similarity, particularly for the two peripheral domains displaying heterogeneity in length and the presence of repeated sequences. The multialignment is available at the WWW site: http://www.ba.cnr.it/dloop.html. Our comparative study has allowed us to identify new conserved sequence blocks present in all the species under consideration and events of insertion/deletion which have important implications in both functional and evolutionary aspects. In particular we have detected two blocks, about 60 bp long, extended termination associated sequences (ETAS1 and ETAS2) conserved in all the organisms considered. Evaluation against experimental work suggests a possible functional role of ETAS1 and ETAS2 in the regulation of replication and transcription and targeted experimental approaches. The analyses on conserved sequence blocks (CSBs) clearly indicate that CSB1 is the only very essential element, common to all mammalian mt genomes, while CSB2 and CSB3 could be involved in different though related functions, probably species specific, and thus more linked to nuclear mitochondrial coevolutionary processes. Our hypothesis on the different functional implications of the conserved elements, CSBs and TASs, reported so far as main regulatory signals, would explain the different conservation of these elements in evolution. Moreover the intra-order comparison of the D-loop regions highlights peculiar features useful to define the evolutionary dynamics of this region in closely related species.

Animals↗

PubMed1999

Structure of the phylogenetically most conserved domain of SRP RNA.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

Base Sequence↗

PubMed2006

Differential O-glycosylation of a conserved domain expressed in murine and human ZP3.

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.

Amino Acid Sequence↗

PubMed2006

Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis.

The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have shown in the past that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD, which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A, and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all of the mutant variants showed characteristics similar to those of the wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. Although the Cdr1p D327N mutant variant showed comparable binding with [alpha-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. The Cdr1p D327N mutant variant showed approximately 40% enhancement of its residual ATPase activity at acidic pH, whereas no such pH effect was seen with the Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.

Amino Acid Sequence↗

PubMed1996

Sequence effects on RNA bulge-induced helix bending and a conserved five-nucleotide bulge from the group I introns.

Bulge loops introduce bends in RNA double helices. Thus, a role for bulge loops in the tertiary folding of RNA is to orient helical elements. The location, size, and sequence of a five-nucleotide bulge are conserved in many of the self-splicing group I introns. We have used gel electrophoretic analysis of helix bending to test the hypothesis that this bulge loop is conserved to control the angle between the flanking helices. Interruption of an RNA duplex by the five-nucleotide bulge of the group I intron from Tetrahymena thermophila results in an electrophoretically retarded species, indicative of bending by the bulge. However, mutation of conserved bases in the bulge has a small effect on the retardation, suggesting that the average induced bend angle is not strongly dependent on the conserved sequence. Electrophoretic analysis of a mixture of bulged duplexes containing all five-nucleotide bulges reveals that most five-nucleotide bulge sequences induce bends that are similar to the bend induced by the conserved bulge. We have calibrated relative electrophoretic mobilities with bends of known magnitude, and characterized the distribution of bulge sequences among bend angles. Though the entire range of bend angles induced by different five-nucleotide bulges is from approximately 45 degrees to 75 degrees, most ( > 85%) five-nucleotide bulge loops induce bends between 65 degrees and 75 degrees. We have identified several of the anomalous five-nucleotide bulge sequences that induce bends of magnitude smaller than 65 degrees. They are generally, though not universally, pyrimidine-rich.

Animals↗