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At least 451 records · Page 25Linked to original sources
PubMed2003

Direct interaction between alpha-actinin and hepatitis C virus NS5B.

It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication. Our results suggest alpha-actinin may be a component of the HCV replication complex.

Actinin↗
PubMed2002

Rho-dependent termination and ATPases in transcript termination.

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA-binding domain (RNA-BD) of known structure as well as an ATP-binding domain (ATP-BD) for which a structure has been proposed based on homology modeling. A model is proposed in which Rho first makes an interaction with a nascent RNA on a C-rich, primarily single-stranded rut region of the transcript as that region emerges from the exit site of RNA polymerase. A subsequent step involves a temporary release of one subunit of the hexamer to allow the 3' segment of the nascent transcript to enter the central channel of the Rho ring. Actions of the Rho structure in the channel on the 3' segment that are coupled to ATP hydrolysis pull the RNA from its contacts with the template and RNA polymerase, thus causing termination of its synthesis.

Adenosine Triphosphatases↗
PubMed2004

Distinct effects of topoisomerase I and RNA polymerase I inhibitors suggest a dual mechanism of nucleolar/nucleoplasmic partitioning of topoisomerase I.

Topoisomerase I is mostly nucleolar, because it plays a preeminent role in ribosomal DNA (rDNA) transcription. It is cleared from nucleoli following exposure to drugs stabilizing covalent DNA intermediates of the enzyme (e.g. camptothecin) or inhibiting RNA polymerases (e.g. actinomycin D), an effect summarily attributed to blockade of rDNA transcription. Here we show that two distinct mechanisms are at work: (i). Both drugs induce inactivation and segregation of the rRNA transcription machinery. With actinomycin D this leads to a co-migration of RNA-polymerase I and topoisomerase I to the nucleolar perimeter. The process has a slow onset (>20 min), is independent of topoisomerase I activity, but requires the N-terminal domain of the enzyme to colocalize with RNA polymerase I. (ii). Camptothecin induces, in addition, immobilization of active topoisomerase I on genomic DNA resulting in rapid nucleolar clearance and spreading of the enzyme to the entire nucleoplasm. This effect is independent of the state of rRNA transcription, involves segregation of topoisomerase I from RNA polymerase I, has a rapid onset (<1 min), and requires catalytic activity but neither the N-terminal domain of topoisomerase I nor its major sumoylation site. Thus, nucleolar/nucleoplasmic partitioning of topoisomerase I is regulated by interactions with RNA polymerase I and DNA but not by sumoylation.

Binding Sites↗
PubMed2006

DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold.

Two RNases, Dicer and Argonaute, are at the heart of the RNA interference (RNAi) molecular machinery responsible for gene silencing. Both RNases contain multiple domains, most of which have been characterized or have functions that can be predicted based on sequence comparisons. However, Dicers of higher eukaryotes contain the domain known as DUF283 which at present has no assigned role. Using sensitive profile-profile comparisons, we detected a divergent double-stranded RNA-binding domain coinciding with the DUF283 of Dicer. This finding has potential implications regarding the mechanistic role of Dicer in RNAi.

Amino Acid Sequence↗
PubMed2006

Characterization of RNA helicase A as component of STAT6-dependent enhanceosome.

Signal transducer and activator of transcription 6 (STAT6) is a regulator of transcription for interleukin-4 (IL-4)-induced genes. The ability of STAT6 to activate transcription depends on functional interaction with other transcription factors and coactivators. We have characterized the mechanism of STAT6-mediated transcriptional activation by identifying STAT6 transcription activation domain (TAD) interacting nuclear proteins. The first of the identified proteins was coactivator protein p100, which regulates IL-4-induced transcription by connecting STAT6 with other transcriptional regulators. Here, we describe RNA helicase A (RHA) as a novel component of STAT6 transcriptosome. In vitro and in vivo experiments indicated that RHA did not directly interact with STAT6, but p100 protein was found to mediate the assembly of the ternary complex of STAT6-p100-RHA. In chromatin immunoprecipitation studies RHA together with p100 enhanced the binding of STAT6 on the human Igepsilon promoter after IL-4 stimulation. RHA enhanced the IL-4-induced transcription, and the participation of RHA in IL-4-regulated transcription was supported by RNAi experiments. Our results suggest that RHA has an important role in the assembly of STAT6 transcriptosome. As RHA is also known to interact with chromatin modifying proteins, the RHA containing protein complexes may facilitate the entry of transcriptional apparatus to the IL-4 responsive promoters.

Animals↗
PubMed2007

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

Adenosine Triphosphatases↗
PubMed2002

Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 A resolution.

The crystal structure of the initiating form of Thermus aquaticus RNA polymerase, containing core RNA polymerase (alpha2betabeta'omega) and the promoter specificity sigma subunit, has been determined at 4 angstrom resolution. Important structural features of the RNA polymerase and their roles in positioning sigma within the initiation complex are delineated, as well as the role played by sigma in modulating the opening of the RNA polymerase active-site channel. The two carboxyl-terminal domains of sigma are separated by 45 angstroms on the surface of the RNA polymerase, but are linked by an extended loop. The loop winds near the RNA polymerase active site, where it may play a role in initiating nucleotide substrate binding, and out through the RNA exit channel. The advancing RNA transcript must displace the loop, leading to abortive initiation and ultimately to sigma release.

Amino Acid Motifs↗
PubMed1993

Marburg virus gene 4 encodes the virion membrane protein, a type I transmembrane glycoprotein.

Gene 4 of Marburg virus, strain Musoke, was subjected to nucleotide sequence analysis. It is 2,844 nucleotides long and extends from genome position 5821 to position 8665 (EMBL Data Library, emnew: MVREPCYC [accession no. Z12132]). The gene is flanked by transcriptional signal sequences (start signal, 3'-UACUUCUUGUAAUU-5'; termination signal, 3'-UAAUUCUUUUU-5') which are conserved in all Marburg virus genes. The major open reading frame encodes a polypeptide of 681 amino acids (M(r), 74,797). After in vitro transcription and translation, as well as expression in Escherichia coli, this protein was identified by its immunoreactivity with specific antisera as the unglycosylated form of the viral membrane glycoprotein (GP). The GP is characterized by the following four different domains: (i) a hydrophobic signal peptide at the amino terminus (1 to 18), (ii) a predominantly hydrophilic external domain (19 to 643), (iii) a hydrophobic transmembrane anchor (644 to 673), and (iv) a small hydrophilic cytoplasmic tail at the carboxy terminus (674 to 681). Amino acid analysis indicated that the signal peptide is removed from the mature GP. The GP therefore has the structural features of a type I transmembrane glycoprotein. The external domain of the protein has 19 N-glycosylation sites and several clusters of hydroxyamino acids and proline residues that are likely to be the attachment sites for about 30 O-glycosidic carbohydrate chains. The region extending from positions 585 to 610 shows significant homology to a domain observed in the envelope proteins of several retroviruses and Ebola virus that has been suspected to be responsible for immunosuppressive properties of these viruses. A second open reading frame of gene 4 has the coding capacity for an unidentified polypeptide 112 amino acids long.

Amino Acid Sequence↗
PubMed2003

Elevated activity of the large form of ADAR1 in vivo: very efficient RNA editing occurs in the cytoplasm.

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.

Adenosine Deaminase↗
PubMed2005

The ASCH superfamily: novel domains with a fold related to the PUA domain and a potential role in RNA metabolism.

Several studies show that transcription coactivators are often bi-functional ribonucleoprotein complexes that also regulate pre-mRNA processing and splicing decisions. Using sensitive sequence profile searches and structural comparisons we show that the C-terminal domain of the human coactivator protein ASC-1 defines a novel superfamily, the ASC-1 homology (ASCH) domain. The approximately 110 amino acid long ASCH domains are widely represented in all the three superkingdoms of life and several prokaryotic viruses. We show that the ASCH superfamily adopts a beta-barrel fold similar to the PUA domain superfamily. Using multiple lines of evidence, we suggest that members of the ASCH superfamily are likely to function as RNA-binding domains in contexts related to coactivation, RNA-processing and possibly prokaryotic translation regulation. Structural analysis of ASCH domains reveals the presence of a potential RNA-binding cleft associated with a conserved sequence motif, which is characteristic of this superfamily. Despite their similar structure, the ASCH and PUA domains appear to occupy distinct functional niches, with the former domains typically occurring in a standalone form in polypeptides, and the latter domains showing fusions to a variety of RNA-modifying enzymes.

Animals↗
PubMed2002

Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris.

A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris. The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes a polypeptide of 977 amino acid residues. The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein. Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A. thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon. Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts. This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid-encoded genes. We have designated this protein NsRpoTp.

Amino Acid Sequence↗
PubMed2005

Structure determination of protein/RNA complexes by NMR.

Structure determination of protein?RNA complexes in solution provides unique insights into factors that are involved in protein/RNA recognition. Here, we review the methodology used in our laboratory to overcome the challenges of protein?RNA structure determination by nuclear magnetic resonance (NMR). We use as two examples complexes recently solved in our laboratory, the nucleolin RBD12/b2NRE and Rnt1p dsRBD/snR47h complexes. Topics covered are protein and RNA preparation, complex formation, identification of the protein/RNA interface, protein and RNA resonance assignment, intermolecular NOE assignment, and structure calculation and analysis.

Magnetic Resonance Spectroscopy↗
PubMed2006

Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.

Active Transport, Cell Nucleus↗
PubMed1999

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo.

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.

Bacterial Proteins↗
PubMed2003

Multiple interactions involving the amino-terminal domain of yeast mtRNA polymerase determine the efficiency of mitochondrial protein synthesis.

The amino-terminal domain (ATD) of Saccharomyces cerevisiae mitochondrial RNA polymerase has been shown to provide a functional link between transcription and post-transcriptional events during mitochondrial gene expression. This connection is mediated in large part by its interactions with the matrix protein Nam1p and, based on genetic phenotypes, the mitochondrial membrane protein Sls1p. These observations led us to propose previously that mtRNA polymerase, Nam1p, and Sls1p work together to coordinate transcription and translation of mtDNA-encoded gene products. Here we demonstrate by specific labeling of mitochondrial gene products in vivo that Nam1p and Sls1p indeed work together in a pathway that is required globally for efficient mitochondrial translation. Likewise, mutations in the ATD result in similar global reductions in mitochondrial translation efficiency and sensitivity to the mitochondrial translation inhibitor erythromycin. These data, coupled with the observation that the ATD is required to co-purify Sls1p in association with mtDNA nucleoids, suggest that efficient expression of mtDNA-encoded genes in yeast involves a complex series of interactions that localize active transcription complexes to the inner membrane in order to coordinate translation with transcription.

Blotting, Southern↗
PubMed1999

Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

Alkylation↗