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A Sentenac

Publications and source records attributed to A Sentenac.

At least 19 recordsLinked to original sources
PubMed2002

Scattering from rough inhomogeneous media: splitting of surface and volume scattering.

The intensity scattered by particles randomly placed beneath a rough interface is studied with rigorous simulations. It is shown that the angular intensity pattern is close to that obtained by adding the intensity scattered by particles under a flat surface to that scattered by a rough homogeneous surface whose permittivity is evaluated with an effective-medium theory. This heuristic splitting rule is accurate for a large range of parameters that are well beyond any perturbative treatment.

Journal Article↗
PubMed2001

Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae.

Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Amino Acid Sequence↗
PubMed2001

High-mobility-group proteins NHP6A and NHP6B participate in activation of the RNA polymerase III SNR6 gene.

Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.

DNA-Binding Proteins↗
PubMed2000

The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3.

RNA polymerase I (Pol I) is dedicated to transcription of the large ribosomal DNA (rDNA). The mechanism of Pol I recruitment onto rDNA promoters is poorly understood. Here we present evidence that subunit A43 of Pol I interacts with transcription factor Rrn3: conditional mutations in A43 were found to disrupt the transcriptionally competent Pol I-Rrn3 complex, the two proteins formed a stable complex when co-expressed in Escherichia coli, overexpression of Rrn3 suppressed the mutant phenotype, and A43 and Rrn3 mutants showed synthetic lethality. Consistently, immunoelectron microscopy data showed that A43 and Rrn3 co-localize within the Pol I-Rrn3 complex. Rrn3 has several protein partners: a two-hybrid screen identified the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by affinity chromatography. Our results suggest that Rrn3 plays a central role in Pol I recruitment to rDNA promoters by bridging the enzyme to the core factor. The existence of mammalian orthologues of A43 and Rrn3 suggests evolutionary conservation of the molecular mechanisms underlying rDNA transcription in eukaryotes.

Amino Acid Sequence↗
PubMed2000

A novel subunit of yeast RNA polymerase III interacts with the TFIIB-related domain of TFIIIB70.

There is limited information on how eukaryotic RNA polymerases (Pol) recognize their cognate preinitiation complex. We have characterized a polypeptide copurifying with yeast Pol III. This protein, C17, was found to be homologous to a mammalian protein described as a hormone receptor. Deletion of the corresponding gene, RPC17, was lethal and its regulated extinction caused a selective defect in transcription of class III genes in vivo. Two-hybrid and coimmunoprecipitation experiments indicated that C17 interacts with two Pol III subunits, one of which, C31, is important for the initiation reaction. C17 also interacted with TFIIIB70, the TFIIB-related component of TFIIIB. The interaction domain was found to be in the N-terminal, TFIIB-like half of TFIIIB70, downstream of the zinc ribbon and first imperfect repeat. Although Pol II similarly interacts with TFIIB, it is notable that C17 has no similarity to any Pol II subunit. The data indicate that C17 is a novel specific subunit of Pol III which participates together with C34 in the recruitment of Pol III by the preinitiation complex.

Amino Acid Sequence↗
PubMed2000

Dipole radiation into grating structures

We present a detailed electromagnetic analysis for the radiation of an electric source located inside grating structures. Our analysis is based on the differential method and uses the scattering-matrix algorithm. We show that gratings that exhibit periodic modulations along two spatial directions (crossed gratings) enable one to couple out the totality of the light emitted by the source into the guided modes of the structure. This property is investigated through the computation of the far-field radiation patterns for crossed gratings with various etching depths. One key result is the possibility to confine the emitted light in a direction about the sample normal, a property that is of interest in the context of spontaneous emission control by microcavity structures.

Journal Article↗
PubMed1999

Interaction between yeast RNA polymerase III and transcription factor TFIIIC via ABC10alpha and tau131 subunits.

Yeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function. Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, tau131, and ABC10alpha, a small essential subunit common to the three forms of nuclear RNA polymerases. This interaction was mapped to the C-terminal region of ABC10alpha. A thermosensitive mutation in the C terminus region of ABC10alpha (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of tau131 (tau131-DeltaTPR2) that lacks the second TPR repeat. Remarkably, the rpc10-30 mutation weakened the ABC10alpha-tau131 interaction, and the suppressive mutation, tau131-DeltaTPR2 increased the interaction between the two proteins in the two-hybrid assay. These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III.

Alleles↗
PubMed1999

Rpb4p is necessary for RNA polymerase II activity at high temperature.

Rpb4p and Rpb7p are two subunits of the yeast RNA polymerase II, which form a subcomplex that can dissociate from the enzyme in vitro. Whereas RPB7 is essential, RPB4 is dispensable for cellular viability. However, the rpb4 null mutant is heat-sensitive, and it has been suggested that Rpb4p is an essential component for cellular stress response. To examine this hypothesis, we used two-dimensional gel electrophoresis to analyze the protein expression pattern of the rpb4 null mutant in response to heat shock, oxidative stress, osmotic stress, and in the post-diauxic phase. We show that this mutant is not impaired in stress induced transcriptional activation: the absence of heat shock response of the mutant is due to a general defect in RNA polymerase II activity at high temperature. Under this condition, Rpb4p is necessary to maintain the polymerase activity in vivo. The heat growth defect of the rpb4 null mutant can be partially suppressed by overexpression of RPB7, suggesting that Rpb4p maintains or stabilizes Rpb7p in the RNA polymerase. We also demonstrate that rpb4 null mutant is an appropriate tool to analyze the involvement of transcriptional events in the survival and adaptation to heat shock or other stresses.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1999

A protein-protein interaction map of yeast RNA polymerase III.

The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

Binding Sites↗
PubMed1999

Mutagenesis of yeast TFIIIB70 reveals C-terminal residues critical for interaction with TBP and C34.

The yeast TFIIIB transcription factor is composed of three components, TBP, TFIIIB90 or B", and TFIIIB70 or BRF. TFIIIB70 is a pivotal component since it interacts with TBP, TFIIIC and RNA polymerase III (pol III). In order to better understand the role of TFIIIB70, we mutagenized extensively three evolutionary conserved motifs of its pol III-specific C-terminal extension. Conditional mutations lying in conserved regions II and III were obtained, some of which altered the interaction with the C34 subunit of pol III and were co-lethal with rpc34 mutations. Two conditional mutations in region II impaired the interaction with TBP and were suppressed by its overexpression. The pattern of suppression of the strongest mutation by overexpression of various mutant TBP, suggested a contact between TBP-R220 and TFIIIB70-D464 residues in vivo. As expected, this TFIIIB70 mutation impaired the assembly of TFIIIB. TFIIIC.DNA complexes and affected in vitro transcription of the SUP4 tRNA gene. Our results underscore the important role of region II of TFIIIB70 in pre-initiation as well as transcription complex assembly via C34 and TBP binding.

Amino Acid Sequence↗
PubMed1999

A subunit of yeast TFIIIC participates in the recruitment of TATA-binding protein.

TFIIIC plays a key role in nucleating the assembly of the initiation factor TFIIIB on class III genes. We have characterized an essential gene, TFC8, encoding the 60-kDa polypeptide, tau60, present in affinity-purified TFIIIC. Hemagglutinin-tagged variants of tau60 were found to be part of TFIIIC-tDNA complexes and to reside at least in part in the downstream DNA-binding domain tauB. Unexpectedly, the thermosensitive phenotype of N-terminally tagged tau60 was suppressed by overexpression of tau95, which belongs to the tauA domain, and by two TFIIIB components, TATA-binding protein (TBP) and B"/TFIIIB90 (but not by TFIIIB70). Mutant TFIIIC was deficient in the activation of certain tRNA genes in vitro, and the transcription defect was selectively alleviated by increasing TBP concentration. Coimmunoprecipitation experiments support a direct interaction between TBP and tau60. It is suggested that tau60 links tauA and tauB domains and participates in TFIIIB assembly via its interaction with TBP.

DNA-Binding Proteins↗
PubMed1998

The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination.

Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit, named C11, that is conserved in humans and shows a strong homology to TFIIS. A mutant Pol III, heterocomplemented with Schizosaccharomyces pombe C11, was affected in transcription termination in vivo. A purified form of the enzyme (Pol III Delta), deprived of C11 subunit, initiated properly but ignored pause sites and was defective in termination. Remarkably, Pol III Delta lacked the intrinsic RNA cleavage activity of complete Pol III. In vitro reconstitution experiments demonstrated that Pol III RNA cleavage activity is mediated by C11. Mutagenesis in C11 of two conserved residues, which are critical for the TFIIS-dependent cleavage activity of Pol II, is lethal. Immunoelectron microscopy data suggested that C11 is localized on the mobile thumb-like stalk of the polymerase. We propose that C11 allows the enzyme to switch between an RNA elongation and RNA cleavage mode and that the essential role of the Pol III RNA cleavage activity is to remove the kinetic barriers to the termination process. The integration of TFIIS function into a specific Pol III subunit may stem from the opposite requirements of Pol III and Pol II in terms of transcript length and termination efficiency.

Amino Acid Sequence↗
PubMed1998

Tau91, an essential subunit of yeast transcription factor IIIC, cooperates with tau138 in DNA binding.

Transcription factor IIIC (TFIIIC) (or tau) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, tau91, present in affinity-purified TFIIIC. Tau91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of tau91 was established by the following biochemical and genetic evidence. Antibodies to tau91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to tau91; and a point mutation in TFC6 (tau91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349E mutant affected in the B block binding subunit (tau138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that tau91 cooperates with tau138 for DNA binding. Recombinant tau91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

Amino Acid Sequence↗
PubMed1998

A chimeric subunit of yeast transcription factor IIIC forms a subcomplex with tau95.

The multisubunit yeast transcription factor IIIC (TFIIIC) is a multifunctional protein required for promoter recognition, transcription factor IIIB recruitment, and chromatin antirepression. We report the isolation and characterization of TFC7, an essential gene encoding the 55-kDa polypeptide, tau55, present in affinity-purified TFIIIC. tau55 is a chimeric protein generated by an ancient chromosomal rearrangement. Its C-terminal half is essential for cell viability and sufficient to ensure TFIIIC function in DNA binding and transcription assays. The N-terminal half is nonessential and highly similar to a putative yeast protein encoded on another chromosome and to a cyanobacterial protein of unknown function. Partial deletions of the N-terminal domain impaired tau55 function at a high temperature or in media containing glycerol or ethanol, suggesting a link between PolIII transcription and metabolic pathways. Interestingly, tau55 was found, together with TFIIIC subunit tau95, in a protein complex which was distinct from TFIIIC and which may play a role in the regulation of PolIII transcription, possibly in relation to cell metabolism.

Amino Acid Sequence↗
PubMed1997

Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers.

To probe the complex nucleic acid binding domains of yeast RNA polymerase II (Pol II), we have isolated in the presence of heparin RNA molecules that selectively bind to yeast Pol II. A class of RNA molecules was found to bind and strongly interfere with enzyme-DNA interaction but not with RNA chain elongation. Remarkably, one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II. S. cerevisiae Pol I and Pol III and Pol II from Schizosaccharomyces pombe or wheat germ cells were not affected. Photocross-linking experiments showed that the RNA ligand preferentially interacted with B220, the largest subunit of Pol II and, to a lesser extent, with B150, the second largest subunit. The selected RNA was expressed in yeast cells under the control of a Pol III promoter. Yeast cells that expressed the anti-Pol II aptamer grew normally. However, a cell growth defect was observed when expressing the RNA aptamer in cells having an artificially reduced level of Pol II.

Base Sequence↗
PubMed1997

Dual role of the C34 subunit of RNA polymerase III in transcription initiation.

The C34 subunit of yeast RNA polymerase (pol) III is part of a subcomplex of three subunits which have no counterpart in the other two nuclear RNA polymerases. This subunit interacts with TFIIIB70 and is therefore thought to participate in pol III recruitment. To study the role of C34 in transcription, we have mutagenized RPC34, the gene encoding C34, and found that mutations affecting growth also altered C34 interaction with TFIIIB70. The two mutant pol III that were purified had catalytic properties indistinguishable from those of the wild-type pol III on a poly[d(A-T)] template, while specific transcription of pol III genes in the presence of general transcription factors was impaired. The defect of the C34-1124 mutant enzyme could be compensated by increasing the amount of pol III present in the reaction, suggesting that the enzyme had a lower affinity for pre-initiation complexes. In contrast, the C34-1109 mutant enzyme was defective in transcription initiation due to impaired open complex formation. These observations demonstrate that the C34 subunit is a major determinant in pol III recruitment by the pre-initiation complex and further acts at a subsequent stage that involves the configuration of an initiation-competent form of RNA polymerase.

Amino Acid Sequence↗