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PubMed2000

Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney.

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations. (1) Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na(+)-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is approximately 24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant (K(m)) for succinate was 0.35 mM, and the K(m) for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at -50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.

Animals↗
PubMed2002

Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues.

Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2,2,7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7)GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G- and m(3)(2,2,7)G-containing caps (K(as) 2 x 10(6) M(-1) to 7 x 10(6) M(-1)) whereas IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K(as) on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.

Animals↗
PubMed2006

Structural basis for non-catalytic and catalytic activities of ribonuclease III.

Ribonuclease III (RNase III) represents a highly conserved family of double-stranded (ds) RNA-specific endoribonucleases, exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. Bacterial RNase III, containing an endonuclease domain followed by a dsRNA-binding domain, is the most extensively studied member of the family. It can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves dsRNA or as a binding protein that binds but does not cleave dsRNA. The available biochemical and structural data support the existence of two distinct forms of the RNase III-dsRNA complex which reflect the dual activities of the protein. The information revealed by the structures of bacterial RNase III provides insight into the mechanism of dsRNA processing by all members of the family.

Animals↗
PubMed2005

Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations.

Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P'-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

Binding Sites↗
PubMed2006

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor.

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

Alanine↗
PubMed1987

Antibody-nucleic acid interactions. Antibodies to psoralen-modified RNA as probes of RNA structure.

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.

Animals↗
PubMed1993

Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities.

RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.

Blotting, Western↗
PubMed2002

Different substitutions at conserved amino acids in domains II and III in the Sendai L RNA polymerase protein inactivate viral RNA synthesis.

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I-VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.

Amino Acid Sequence↗
PubMed2005

Selective ablation of retinoblastoma protein function by the RET finger protein.

The retinoblastoma tumor suppressor protein (Rb) affects gene transcription both negatively and positively and through this regulates distinct cellular responses. Although cell cycle regulation requires gene repression, Rb's ability to promote differentiation and part of its antiproliferative activity appears to rely on the activation of gene transcription. We present evidence here that the RET finger protein (RFP)/tripartite motif protein 27 (TRIM 27) inhibits gene transcription activation by Rb but does not affect gene repression. RFP binds to Rb and prevents the degradation of the EID-1 inhibitor of histone acetylation and differentiation. Furthermore, ablation of RFP in U2OS osteosarcoma cells augments a transcriptional program indicative of lineage-specific differentiation in response to Rb. These findings provide precedent for a regulatory pathway that uncouples different Rb-dependent activities and thus silences specific cellular responses to Rb in a selective way.

Adenovirus E1A Proteins↗
PubMed2003

Loading Rho to terminate transcription.

In bacteria, one of the major transcriptional termination mechanisms requires a hexameric RNA/DNA helicase known as Rho. One question that has remained unanswered is how the helicase loads onto a nascent transcript so that it can initiate actions on the transcript to cause termination. Recent structures of Rho bound to nucleic acid by show how the individual RNA-binding domains of the 6 subunits are organized and that the ring is split open. The opening is wide enough to accommodate single-stranded RNA and suggests that this conformation is poised to load onto mRNA.

Binding Sites↗
PubMed2001

Periodic perturbations in Shaker K+ channel gating kinetics by deletions in the S3-S4 linker.

Upon depolarization positive charges contained in the transmembrane segment S4 of voltage-dependent channels are displaced from the cytoplasmic to the external milieu. This charge movement leads to channel opening. In Shaker K+ channels four positively charged arginines in the S4 domain are transferred from the internal to the external side of the channel during activation. The distance traveled by the S4 segment during activation is unknown, but large movements should be constrained by the S3-S4 linker. Constructing deletion mutants, we show that the activation time constant and the midpoint of the voltage activation curve of the Shaker K+ channel macroscopic currents becomes a periodic function of the S3-S4 linker length for linkers shorter than 7 aa residues. The periodicity is that typical of alpha-helices. Moreover, a linker containing only 3 aa is enough to recover the wild-type phenotype. The deletion method revealed the importance of the S3-S4 linker in determining the channel gating kinetics and indicated that the alpha-helical nature of S4 extends toward its N terminus. These results support the notion that a small displacement of the S4 segment suffices to displace the four gating charges involved in channel opening.

Animals↗
PubMed2001

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines.

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases.

Apoptosis↗
PubMed2005

Phylogenetic analysis of eIF4E-family members.

BACKGROUND: Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m7Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the 7-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other. RESULTS: By combining sequence data deposited in the Genbank databases, we have identified sequences encoding 411 eIF4E-family members from 230 species. These sequences have been deposited into an internet-accessible database designed for sequence comparisons of eIF4E-family members. Most members can be grouped into one of three classes. Class I members carry Trp residues equivalent to Trp-43 and Trp-56 of H. sapiens eIF4E and appear to be present in all eukaryotes. Class II members, possess Trp-->Tyr/Phe/Leu and Trp-->Tyr/Phe substitutions relative to Trp-43 and Trp-56 of H. sapiens eIF4E, and can be identified in Metazoa, Viridiplantae, and Fungi. Class III members possess a Trp residue equivalent to Trp-43 of H. sapiens eIF4E but carry a Trp-->Cys/Tyr substitution relative to Trp-56 of H. sapiens eIF4E, and can be identified in Coelomata and Cnidaria. Some eIF4E-family members from Protista show extension or compaction relative to prototypical eIF4E-family members. CONCLUSION: The expansion of sequenced cDNAs and genomic DNAs from all eukaryotic kingdoms has revealed a variety of proteins related in structure to eIF4E. Evolutionarily it seems that a single early eIF4E gene has undergone multiple gene duplications generating multiple structural classes, such that it is no longer possible to predict function from the primary amino acid sequence of an eIF4E-family member. The variety of eIF4E-family members provides a source of alternatives on the eIF4E structural theme that will benefit structure/function analyses and therapeutic drug design.

Amino Acid Sequence↗
PubMed1993

Modeling the three-dimensional structure of RNA.

The limited number of RNA structures determined by X-ray crystallography and NMR spectroscopy compels the use of experimental and theoretical methods that are less precise to obtain information on RNA conformation. RNA flexibility, a consequence of rotational freedom about seven intra- and internucleotide bonds, is unfortunately of such magnitude that these alternate techniques fall short of providing sufficient information to build robust tertiary structures. Various RNA modeling methods, described herein, permit the organization of this structural data to the form of three-dimensional structures. Interactive computer graphics techniques, for example, have generated several useful models. Also, conventional computer algorithms involving the minimization of empirical energy functions, previously limited to small molecules, are giving way to methods able to handle much larger molecules. Modified distance geometry and molecular mechanics algorithms, using simplified "pseudoatom" representations, can generate structures consistent with input data. A constraint satisfaction algorithm combined with discrete representations of nucleotide conformations systematically explores poorly defined regions of a molecule yielding all-atom representations, but requires enough structural constraints to avoid a computational explosion.

Algorithms↗
PubMed2005

Purified Argonaute2 and an siRNA form recombinant human RISC.

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

Adenosine Triphosphate↗
PubMed2003

Determinants within an 18-amino-acid U1A autoregulatory domain that uncouple cooperative RNA binding, inhibition of polyadenylation, and homodimerization.

The human U1 snRNP-specific U1A protein autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. Previous work demonstrated that a short sequence of U1A protein is essential for autoregulation and contains three distinct activities, which are (i) cooperative binding of two U1A proteins to a 50-nucleotide region of U1A pre-mRNA called polyadenylation-inhibitory element RNA, (ii) formation of a novel homodimerization surface, and (iii) inhibition of polyadenylation by inhibition of poly(A) polymerase (PAP). In this study, we purified and analyzed 11 substitution mutant proteins, each having one or two residues in this region mutated. In 5 of the 11 mutant proteins, we found that particular amino acids associate with one activity but not another, indicating that they can be uncoupled. Surprisingly, in three mutant proteins, these activities were improved upon, suggesting that U1A autoregulation is selected for suboptimal inhibitory efficiency. The effects of these mutations on autoregulatory activity in vivo were also determined. Only U1A and U170K are known to regulate nuclear polyadenylation by PAP inhibition; thus, these results will aid in determining how widespread this type of regulation is. Our molecular dissection of the consequences of conformational changes within an RNP complex presents a powerful example to those studying more complicated pre-mRNA-regulatory systems.

Amino Acid Sequence↗
PubMed2006

Structural basis for double-stranded RNA processing by Dicer.

The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.

Amino Acid Sequence↗