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A Sentenac

Publications and source records attributed to A Sentenac.

At least 91 records · Page 5Linked to original sources
PubMed1990

Mapping the active site of yeast RNA polymerase B (II).

Yeast RNA polymerase B (II) was incubated with a collection of 13 different nucleotide derivatives and affinity labeled by allowing DNA-directed phosphodiester bond formation. The 32P-labeled site was localized in the C-terminal part of the B150 subunit by microsequencing a proteolytic fragment, then further mapped by a combination of extensive or single-hit chemical cleavage reactions and analysis of the labeled peptide patterns. The affinity label was mapped to between Asn946 and Met999, within one of the nine regions that are conserved between B150 and the bacterial beta subunit. The results underscore the conservative evolution of the catalytic center of eukaryotic and bacterial RNA polymerases.

Amino Acid Sequence↗
PubMed1990

ABF1 binding sites in yeast RNA polymerase genes.

We have used gel retardation and DNase protection assays to investigate the trans-acting factors involved in the regulation of yeast RNA polymerase genes RPC160 and RPC40. The same binding component was found to interact with the promoter of the two genes, at a short distance (100-150 base pairs) from the transcription start sites. From its size, its DNA-binding specificity and its immunological properties, this factor appears to correspond to the autonomous replication sequences and silencer-binding factor ABF1/SBF-B. The interaction of ABF1 with the polymerase upstream box sequence was characterized using gel DNA-binding assay. The factor binds with high affinity to the polymerase upstream box sequence (Kapp = 5.10(-10) M). A mutational analysis showed that nine base pairs belonging to two separated attachment sites are involved in factor binding. The consensus sequence RTCRYB(N)4ACG was derived from the present binding studies. These data provide an experimental basis for evaluating the efficiency of known or potential ABF1 sites and for comparing several factors with ABF1-like binding properties.

Amino Acid Sequence↗
PubMed1990

A yeast homolog of the human UEF stimulates transcription from the adenovirus 2 major late promoter in yeast and in mammalian cell-free systems.

We report the identification and purification of a yeast factor functionally homologous to the human upstream element factor (UEFh). Although the yeast protein (UEFy) has a higher molecular weight than the HeLa UEF (60 kD versus 45 kD) both have identical DNA-binding properties: the purified UEFy recognizes the Adenovirus 2 (Ad2) major late promoter upstream element (MLP-UE; from nucleotide -49 to -67) as well as the IVa2 upstream element (IVa2-UE; from nucleotide -98 to -122) with a higher affinity for the MLP-UE than for the IVa2-UE. Based on its DNA binding specificity, size and thermostability, the UEFy protein appears also similar or equivalent to the centromere binding protein CP1. In a competition assay with oligonucleotides containing the MLP-UE binding site, a drastic reduction of Ad2 MLP transcription was observed both in a HeLa and in a yeast cell free system, which was restored by addition of either the purified UEFh or UEFy proteins. We conclude that both UEFh and UEFy activate transcription from the Ad2 MLP upon binding to the upstream element, whatever is the in vitro cell-free system (yeast or HeLa). This indicate that some regulatory function represented by the upstream element and its cognate factor, is well conserved between human and yeast.

Adenoviruses, Human↗
PubMed1990

Contacts between the factor TUF and RPG sequences.

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

Base Sequence↗
PubMed1990

On the flexible interaction of yeast factor tau with the bipartite promoter of tRNA genes.

Yeast transcription factor tau (analogous to vertebrate TFIIIC) interacts specifically with the internal split promoter of tRNA genes. Binding to the two promoter elements (A block and B block) occurs within 30 seconds even when they are separated by a long intervening sequence. Dimethylsulfate protection analysis of contact points between tau and the noncoding strand of a series of internally deleted tRNA3(Leu) genes shows that the specificity of the interaction is not affected by changes in the distance or in the relative helical orientation of the promoter elements. This result is consistent with the results of previous footprinting experiments (Baker, R.E., Camier, S., Sentenac, A. and Hall, B.D., 1987, Proc. Natl. Acad. Sci. USA, 84, 8768-8772). To test if any physical constraint is imposed on the DNA molecule upon tau binding, we analyzed the effect of introducing random single-strand breaks in the noncoding strand of the tRNA gene. Whereas some nicks located in the A block were found to prevent tau binding, no single-strand break in the B block region or in the DNA between the A and B blocks were observed to inhibit or facilitate the binding of tau. We therefore propose that the great flexibility of the tau-tDNA interaction is mostly due to the tau protein itself.

DNA, Fungal↗
PubMed1990

The U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C (III) in vivo and in vitro.

Unlike the majority of genes encoding small nuclear RNAs, which are transcribed by RNA polymerase B, the U6 gene contains features found in both class B and class C genes, indicating the involvement of a combination of transcription factors normally specific to each class of genes. We present direct genetic and biochemical evidence that the U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C in vivo as well as in vitro. A mutant strain with a temperature-sensitive defect in the large subunit of RNA polymerase C that results in defective transcription of tRNA and 5S RNA genes shows a corresponding defect in U6 RNA levels. Also, purified RNA polymerase C transcribes the U6 gene when supplemented with partially purified TFIIIB. The other class C transcription factors, TFIIIA and Tau (TFIIIC), are not required in this system.

Base Sequence↗
PubMed1990

Electron microscopic study of yeast RNA polymerase A: analysis of single molecular images.

The structural features of the yeast DNA-dependent RNA polymerase A (I) were examined by Scanning Transmission Electron Microscopy. The enzyme was absorbed in its monomeric form and negatively stained prior to digital image acquisition at low dose. The signal to noise ratio of single particle images was improved through averaging of a large number of previously aligned and partitioned images. Six classes of images were obtained reproducibly which corresponded to different projections of the enzyme. The enzyme structure was characterized by its presence of two curved arms which defined a longitudinal cleft. By analogy with the Escherichia coli enzyme, these arms could correspond to the two large subunits A135 and A190.

Chemical Phenomena↗
PubMed1990

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae.

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

Base Sequence↗
PubMed1990

The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail.

The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library. It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site. Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31. It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W. Haggren and D. Kolodrubetz, Mol. Cell. Biol. 8:1282-1289, 1988). Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C. The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1. Outside this domain, however, there is no appreciable homology to any known protein. The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function. Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31. Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C.

Amino Acid Sequence↗
PubMed1989

The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy.

Yeast transcription factor tau interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified tau factor and tau-tDNA complexes were examined by scanning transmission electron microscopy to analyze the structural features of free and DNA bound factor. The free factor appeared as two tightly associated globular domains of roughly similar size (10 nm in diameter) and mass (approximately 300 kd). A combination of these two domains results in a mass for the factor of 510-670 kd. When tau was allowed to interact with recombinant tRNA(3Leu) genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and tau appeared as a large particle covering the A and B block region. On the other hand, with genes having a larger A-B distance (53 or 74 bp), mostly dumb-bell-shaped complexes were formed with individualized factor domains bound separately to the A and B blocks. A smaller proportion of the complexes appeared to consist of a large particle bound at only one site, essentially on the B block. Mapping of the binding domains in the DNA showed a good correlation with the respective positions of the A and B promoter elements. Factor binding did not induce a noticeable DNA bending, although with extended genes apparent DNA shortening and cases of DNA looping were observed. Upon cleavage of the tRNA(3Leu) gene between the A and B blocks after or prior to complex formation, the two factor domains remained attached to the same DNA fragment (mostly the B-DNA fragment). In addition, images of protein-linked, reconstituted full-length genes were also observed. These different conformational states of the tau-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.

DNA Polymerase III↗
PubMed1989

Asymmetric DNA bending induced by the yeast multifunctional factor TUF.

TUF is a yeast regulatory factor that binds to conserved DNA sequence elements involved in gene activation or silencing as well as in telomere function. Using gel electrophoresis analyses, we show here that TUF induces DNA bending at a site located upstream of the recognition sequence (rpg box). Several point mutations in the rpg box reduced TUF binding strength without affecting the extent of bending. Selective proteolysis of TUF.DNA complexes further suggested the existence of two separate protein domains involved in DNA bending and specific DNA recognition. DNA bending may be an important feature of multifunctional factors that could help them to recruit other proteins for the formation of multiprotein complexes.

Cloning, Molecular↗
PubMed1989

The yeast H+-ATPase gene is controlled by the promoter binding factor TUF.

The H+-ATPase, located in the yeast plasma membrane and encoded by the PMA1 gene, provides energy for the active transport of nutrients and regulates intracellular pH. Expression of the PMA1 gene is essential for cell growth and development. In this study, progressive deletions of the PMA1 promoter fused to the beta-galactosidase gene have identified two upstream activating sequences. These upstream activating sequences have high homologies with the consensus sequence known to control the expression of the ribosomal protein genes (RPG). In vivo deletion of these RPG sequences from the PMA1 gene results in slower growth and reduces ATPase activity to one-third of its original value. The RPG sequences from PMA1 interact with the promoter binding factor TUF. Thus, PMA1 belongs to the RPG-TUF system which includes many constitutive genes encoding nonrelated functions such as ATP metabolism, transcription, translation, and active transport.

DNA Mutational Analysis↗
PubMed1989

Two polypeptide chains in yeast transcription factor tau interact with DNA.

Yeast transcription factor tau interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of tau was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with tau-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of tau-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to 32P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor tau, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of tau generated a protease-resistant tau B (tau B) domain that binds solely to the B block. tau B-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to tau B and is responsible for B block binding.

Antigen-Antibody Reactions↗
PubMed1988

RNA polymerases B and C are more closely related to each other than to RNA polymerase A.

Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements.

Amino Acid Sequence↗
PubMed1988

A yeast activity can substitute for the HeLa cell TATA box factor.

Most class B (II) promoter regions from higher eukaryotes contain the TATA box and upstream and enhancer elements. Both the upstream and enhancer elements and their cognate factors have regulatory functions, whereas the TATA sequence interacts with the TATA box factor BTF1 to position RNA polymerase B and its ancillary initiation factors (STF, BTF2 and BTF3) to direct the initiation of transcription approximately 30 base pairs downstream. In many respects, class B promoter regions from the unicellular eukaryote Saccharomyces cerevisiae are similarly organized, containing upstream activating sequences that bear many similarities to enhancers. Although they are essential for initiation, the yeast TATA sequences are located at variable distances and further from the start sites (40-120 base pairs), whose locations are primarily determined by an initiator element. The basic molecular mechanisms that control initiation of transcription are known to be conserved from yeast to man: the yeast transcriptional transactivator GAL4 can activate a minimal TATA box-containing promoter in human HeLa cells, and a human inducible enhancer factor, the oestrogen receptor, can activate a similar minimal promoter in yeast. This striking evolutionary conservation prompted us to look for the presence in yeast of an activity that could possibly substitute for the human TATA box factor. We report here the existence of such an activity in yeast extracts.

Base Sequence↗
PubMed1988

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A.

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.

Amino Acid Sequence↗
PubMed1988

Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae.

A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

Chromosome Mapping↗