Showing papers on "Upstream activating sequence published in 1990"

The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.

Abstract: The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.

Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter.

Abstract: The c-myc protooncogene has been implicated in control of growth and differentiation of mammalian cells. For instance, growth arrest is often preceded by reduction in c-myc mRNA and gene transcription. To elucidate the mechanisms of control of c-myc gene transcription, we have begun to characterize the interaction of nuclear factors with the 719-base-pair (bp) c-myc regulatory domain, located 1139-421 bp upstream of the P1 start site of the mouse gene. Nuclear extracts from exponentially growing WEHI 231 murine B-lymphoma cells formed multiple complexes in mobility-shift assays. Changes in complex distribution were observed in growth-arrested WEHI 231 cells, and a major site of this interaction mapped to a 21-bp sequence that is similar to the sequences recognized by the NF-kappa B family of proteins. Binding of NF-kappa B-like factors was demonstrated by oligonucleotide competition. Induction of complex formation upon 70Z/3 pre-B- to B-cell differentiation, enhancement of binding by GTP, and detergent-induced release of inhibitor protein suggested that NF-kappa B itself is one member of the family that can bind. Transfection of thymidine kinase-chloramphenicol acetyltransferase constructs containing the 21-bp c-myc sequence into Jurkat cells demonstrated increased chloramphenicol acetyltransferase activity upon phorbol ester and phytohemagglutinin treatment. These results suggest the involvement of NF-kappa B-like factors in the regulation of c-myc transcription.

A yeast protein that influences the chromatin structure of UASG and functions as a powerful auxiliary gene activator.

Abstract: GRF2, an abundant yeast protein of Mr -127,000, binds to the GAL upstream activating sequence (UASc) and creates a nucleosome-free region of -230 bp. Purified GRF2 binds to sequences found in many other UASs, in the 35S rRNA enhancer, at centromeres, and at telomeres. Although GRF2 stimulates transcription only slightly on its own, it combines with a neighboring weak activator to give as much as a 170-fold enhancement. This effect of GRF2 is strongly distance-dependent, declining by 85% when 22 bp is interposed between the GRF2 and neighboring activator sites.

The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1.4 gene

Abstract: Krox-20 is a mouse zinc finger gene expressed in a segment-specific manner in the early central nervous system, which makes it a potential developmental control gene. In this report, we show that the Krox-20 protein binds in vitro to two specific DNA sites located upstream from the homeobox containing gene Hox-1.4. The nucleotide sequence recognized by Krox-20 is closely related to the Sp1 target sequence, which is consistent with the similarity existing between the zinc fingers of the two proteins. In co-transfection experiments in cultured cells, Krox-20 dramatically activates transcription from the herpes simplex virus thymidine kinase promoter when an oligomer of its binding site is present in cis close to the promoter. Analysis of mutated binding sites demonstrates that the level of activation by Krox-20 correlates with the affinity of the protein for the mutant sequence. These data indicate that Krox-20 constitutes a sequence-specific DNA-binding transcription factor. Parallel analysis of the expression of Krox-20 and Hox-1.4 in the neural tube by in situ hybridization revealed no overlap, arguing against direct interactions between these two genes. The possible involvement of Krox-20 in the regulation of the transcription of other homeobox genes is discussed in view of their respective patterns of expression.

The role of FIS in trans activation of stable RNA operons of E. coli.

Abstract: The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.

RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals.

Abstract: The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus. Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures. Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs. The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme. We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast. The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another. One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor.

A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

Abstract: ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae.

Abstract: To determine whether the 70-kilodalton heat shock proteins of Saccharomyces cerevisiae play a role in regulating their own synthesis, we studied the effect of overexpressing the SSA1 protein on the activity of the SSA1 5'-regulatory region. The constitutive level of Ssa1p was increased by fusing the SSA1 structural gene to the GAL1 promoter. A reporter vector consisting of an SSA1-lacZ translational fusion was used to assess SSA1 promoter activity. In a strain producing approximately 10-fold the normal heat shock level of Ssa1p, induction of beta-galactosidase activity by heat shock was almost entirely blocked. Expression of a transcriptional fusion vector in which the CYC1 upstream activating sequence of a CYC1-lacZ chimera was replaced by a sequence containing a heat shock upstream activating sequence (heat shock element 2) from the 5'-regulatory region of SSA1 was inhibited by excess Ssa1p. The repression of an SSA1 upstream activating sequence by the SSA1 protein indicates that SSA1 self-regulation is at least partially mediated at the transcriptional level. The expression of another transcriptional fusion vector, containing heat shock element 2 and a lesser amount of flanking sequence, is not inhibited when Ssa1p is overexpressed. This suggests the existence of an element, proximal to or overlapping heat shock element 2, that confers sensitivity to the SSA1 protein.

Regulation of a yeast HSP70 gene by a cAMP responsive transcriptional control element.

Abstract: HSP70 genes exhibit complex regulation in response to stress and a variety of cellular and developmental events. The SSA3 HSP70 gene of Saccharomyces cerevisiae is activated at the transcriptional level under conditions of nutrient limitation. Analysis of deletions revealed that cis-acting DNA sequences present immediately upstream and downstream of the previously identified heat shock elements (UASHS) mediate this regulation. A 35 bp region of SSA3, distinct from UASHS, contains sequences capable of activating a heterologous promoter following the diauxic shift and in the stationary phase of the yeast life cycle; this region has been designated an upstream activating sequence, UASPDS. Expression driven by UASPDS is regulated by the RAS/cAMP pathway. Reduced cAMP dependent protein kinase activity results in UASPDS dependent activation of the SSA3 promoter while constitutive cAMP dependent protein kinase activity prevents UASPDS mediated transcription, even under growth conditions that would normally result in full activation. Although the heat shock element alone exhibits no UAS activity under conditions in which UASPDS promotes transcription, UASHS interacts positively with UASPDS to mediate high levels of SSA3 transcription in response to nutrient limitation and lowered intracellular cAMP concentration. This interaction is independent of the precise spacing and relative orientation of the two elements.

Transcription factor Oct-2A contains functionally redundant activating domains and works selectively from a promoter but not from a remote enhancer position in non-lymphoid (HeLa) cells.

Abstract: In non-lymphoid cells such as HeLa cells, ectopic expression of the lymphocyte-specific transcription factor Oct-2A can activate reporter genes whose promoters consist of a single octamer sequence (ATTTGCAT) upstream of a TATA box. While the factor is strongly active in a promoter position, it tails as an enhancer factor: an enhancer consisting of multiple copies of the octamer sequence placed downstream of the reporter gene is not active in HeLa cells, even at high concentration of Oct-2A. In B lymphoid cells, however, the same enhancer is highly active. This could mean that an additional factor is required for enhancer activation in B cells. Furthermore, we have tested the transcriptional activation potential of Oct-2A with a series of N-terminal and C-terminal deletions. We show that a glutamine-rich domain near the N-terminus is required for full activity. Otherwise, large segments of the N-terminal half or the entire C-terminal region are dispensable in our assay, as long as the deletions do not impinge on the conserved POU domain which is sufficient for DNA binding. While N-terminal and C-terminal regions can functionally compensate for each other, a combined deletion that only retains the POU domain is a strong down mutation. We also find that activity depends on the promoter structure of the reporter gene: the POU domain by itself shows some activity with a promoter where the octamer sequence is located very close to the TATA box, but no activity with another promoter construction where the octamer sequence is located further upstream. The two promoters also respond differently to the deletion of the glutamine-rich stretch important for transcriptional activation. From these experiments we consider it likely that the natural octamer factor variants can selectively activate the different naturally occurring octamer-containing promoters.

A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene.

Abstract: A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

cis- and trans-acting regulatory elements of the yeast URA3 promoter.

Abstract: Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

The promoter of the human interleukin-2 gene contains two octamer-binding sites and is partially activated by the expression of Oct-2.

Abstract: The gene encoding interleukin-2 (IL-2) contains a sequence 52 to 326 nucleotides upstream of its transcriptional initiation site that promotes transcription in T cells that have been activated by costimulation with tetradecanoyl phorbol myristyl acetate (TPA) and phytohemagglutinin (PHA). We found that the ubiquitous transcription factor, Oct-1, bound to two previously identified motifs within the human IL-2 enhancer, centered at nucleotides -74 and -251. Each site in the IL-2 enhancer that bound Oct-1 in vitro was also required to achieve a maximal transcriptional response to TPA plus PHA in vivo. Point mutations within either the proximal or distal octamer sequences reduced the response of the enhancer to activation by 54 and 34%, respectively. Because the murine T-cell line EL4 constitutively expresses Oct-2 and requires only TPA to induce transcription of the IL-2 gene, the effect of Oct-2 expression on activation of the IL-2 promoter in Jurkat T cells was determined. Expression of Oct-2 potentiated transcription 13-fold in response to TPA plus PHA and permitted the enhancer to respond to the single stimulus of TPA. Therefore, both the signal requirements and the magnitude of the transcription response of the IL-2 promoter can be modulated by Oct-2.

Mutations that define the optimal half-site for binding yeast GCN4 activator protein and identify an ATF/CREB-like repressor that recognizes similar DNA sites.

Abstract: The yeast GCN4 transcriptional activator protein binds as a dimer to a dyad-symmetric sequence, indicative of a protein-DNA complex in which two protein monomers interact with adjacent half-sites. However, the optimal GCN4 recognition site, ATGA(C/G)TCAT, is inherently asymmetric because it contains an odd number of base pairs and because mutation of the central C.G base pair strongly reduces specific DNA binding. From this asymmetry, we suggested previously that GCN4 interacts with nonequivalent and possibly overlapping half-sites (ATGAC and ATGAG) that have different affinities. Here, we examine the nature of GCN4 half-sites by creating symmetrical derivatives of the optimal GCN4 binding sequence that delete or insert a single base pair at the center of the site. In vitro, GCN4 bound efficiently to the sequence ATGACGTCAT, whereas it failed to bind to ATGAGCTCAT or ATGATCAT. These observations strongly suggest that (i) GCN4 specifically recognizes the central base pair, (ii) the optimal half-site for GCN4 binding is ATGAC, not ATGAG, and (iii) GCN4 is a surprisingly flexible protein that can accommodate the insertion of a single base pair in the center of its compact binding site. The ATGACGTCAT sequence strongly resembles sites bound by the yeast and mammalian ATF/CREB family of proteins, suggesting that GCN4 and the ATF/CREB proteins recognize similar half-sites but have different spacing requirements. Unexpectedly, in the context of the his3 promoter, the ATGACGTCAT derivative reduced transcription below the basal level in a GCN4-independent manner, presumably reflecting DNA binding by a distinct ATF/CREB-like repressor protein. In other promoter contexts, however, the same site acted as a weak upstream activating sequence.

Yeast Gal11 protein mediates the transcriptional activation signal of two different transacting factors, Gal4 and general regulatory factor I/repressor/activator site binding protein 1/translation upstream factor.

Abstract: GAL11 was first identified as a gene required for full expression of some of the galactose-inducible genes in the yeast Saccharomyces cerevisiae. A null mutation within the GAL11 locus causes defects in mating, growth on nonfermentable carbon sources, and sporulation of gal11 homozygotes. The mating defect was observed only in MAT alpha gal11 strains. Northern hybridization analysis revealed that a gal11 mutation impaired transcription of alpha-specific genes (MF alpha 1 and STE3) but not of an a-specific gene (STE2). Furthermore, this mutation reduced expression of the MAT alpha locus, suggesting that a deficiency in MAT alpha 1 protein is responsible for the reduced expression of alpha-specific genes. Since general regulatory factor I (GRFI)/repressor/activator site binding protein 1 (RAP1)/translation upstream factor (TUF) is believed to be an activator of MAT alpha expression, we examined whether PYK1, which is known to be regulated by GRFI/RAP1/TUF, is also affected by the gal11 mutation. It was determined that the level of PYK1 message was significantly lowered by the mutation. The requirement for functional GAL11 in transcriptional activation was bypassed when either the upstream activating sequence of galactose-inducible genes or of PYK1 was placed very close to the TATA box, suggesting that one of the Gal11 protein functions is to mediate the activation signal of Gal4 and GRFI/RAP1/TUF, when the respective binding site is situated at the naturally occurring distance from the TATA box.

A single DNA-binding transcription factor is sufficient for activation from a distant enhancer and/or from a promoter position.

Abstract: Typical cell type-specific or inducible mammalian genes are under the control of one or more remote enhancers which transmit their effect to the promoter region located at the initiation site of transcription. Both enhancers and promoters are composed of multiple binding sites for transcription factors. To study the requirements for promoter and enhancer function, we have used a reporter gene that is completely dependent on a single DNA-binding transcription factor in vivo. This factor is a truncated, hormone-independent form of the glucocorticoid receptor which interacts strongly with a palindromic binding site. After transfection into HeLa cells, transcription of a reporter gene with one, two or four copies of the binding site upstream of the TATA box is enhanced less than 10, at least 100 and greater than 1000-fold respectively, in the presence of the receptor. Even when the TATA box is deleted, the four upstream binding sites confer receptor-dependent transcription, though from scattered initiation sites. When four copies of the palindromic binding site are placed downstream of the transcription unit, they form a very strong receptor-dependent enhancer. This enhancer can activate comparably well promoters containing binding sites for either glucocorticoid receptor, Sp1 factor, or octamer factor. Our data show that a single defined DNA-binding factor can mediate both promoter and enhancer activity, and that it can co-operate functionally both with itself and with seemingly unrelated transcription factors.

Positive and negative elements regulate human interleukin 3 expression.

Abstract: The human interleukin 3 (IL-3) promoter is comprised of several cis-acting DNA sequences that modulate T-cell expression of IL-3. These are located within 315 nucleotides upstream of the mRNA start site. Transient expression of reporter genes linked to serially deleted sequences of the IL-3 promoter has allowed mapping of two activator sequences and an interposed repressor sequence. The proximal regulatory region is specific to IL-3 and prerequisite for efficient transcription. Its effect is enhanced by a second, more distal activating sequence consisting of an AP-1 binding site. Between the two activators lies a transcriptional silencer, which is a potent repressor in the absence of the AP-1 site. DNA-nuclear protein binding experiments demonstrate specific complex formation within each of these functional regions. Thus, both positive and negative regulatory elements appear to control expression of the human IL-3 gene in activated T cells.

A novel tumor necrosis factor-responsive transcription factor which recognizes a regulatory element in hemopoietic growth factor genes.

Abstract: A conserved DNA sequence element, termed cytokine 1 (CK-1), is found in the promoter regions of many hemopoietic growth factor (HGF) genes. Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor, NF-GMa, which is a protein with a molecular mass of 43 kilodaltons. It interacts with different affinities with the CK-1-like sequence from a number of HGF genes, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF, interleukin 3 (IL-3), and IL-5. We show here that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor-alpha (TNF-alpha) treatment and that the CK-1 sequence from the G-CSF gene is a TNF-alpha-responsive enhancer in these cells. The NF-GMa protein is distinct from another TNF-alpha-responsive transcription factor, NF-kappa B, by several criteria. Firstly, several NF-kappa B-binding sites, although having sequence similarity with the CK-1 sequence, cannot compete efficiently for NF-GMa binding to CK-1. Secondly, the CK-1 sequence from both G-CSF and GM-CSF does not respond to phorbol ester treatment as would an NF-kappa B-binding element. These results demonstrate that NF-GMa is a novel transcription factor inducible by TNF-alpha and binds to a common element in HGF gene promoters.

Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae.

Abstract: Transcription of the DNA damage-responsive gene, DDRA2, of Saccharomyces cerevisiae is activated by heat shock treatment as well as by mutagen/carcinogen exposure. Deletion analysis of upstream noncoding sequence indicated that sequences between approximately -190 and -140 base pairs were necessary for heat shock and DNA damage regulation of transcription. Fusion of this region to a CYC1-lacZ reporter gene demonstrated that the sequence between -202 and -165 base pairs was sufficient for basal level and heat shock-induced expression. This DNA sequence was unable to bind heat shock transcription factor as judged by binding competition experiments in vitro. These results indicate that yeast possesses a second, heat shock transcription factor-independent mechanism for activating transcription in response to thermal stress.

Evidence for multiple OmpR-binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli: a single OmpR-binding site is capable of activating the promoter.

Abstract: We present experimental evidence for the existence of multiple activator-binding sites in the upstream sequence of the ompC promoter, the expression of which is activated by the positive regulator OmpR in response to the osmolarity of the medium. We also found that a single OmpR-binding site can activate the ompC promoter, providing that the binding site is close and placed stereospecifically with respect to the canonical-35 and -10 regions.

Identification of a Saccharomyces cerevisiae DNA-binding protein involved in transcriptional regulation.

Abstract: A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989). When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription. The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene. A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.