Repressor Activator Protein Research Articles

Analysis of the RAP1 protein binding to homogeneous telomeric repeats in Saccharomyces castellii.

The repressor activator protein 1 (RAP1) plays a role in telomere structure and function inS. cerevisiae. Here, the RAP1 homologue was identified and cloned from the budding yeast Saccharomyces castellii (scasRAP1). The scasRAP1 gene encodes a protein of 826 amino acids and shares an overall high degree of similarity with the S. cerevisiae RAP1 (scerRAP1). We demonstrate that the scasRAP1 is able to complement scerRAP1 in temperature-sensitive S. cerevisiae strains and is able to function as a regulator to maintain the original telomere lengths. Binding analyses of the E. coli-expressed scasRAP1 protein demonstrate that it needs two consecutive telomeric repeats in order to bind the S. castellii telomeric DNA sequences, and that it binds adjacent sites having a 16 bp centre-to-centre spacing. The binding affinity to telomeric DNA of several other yeasts is similar to that of scerRap1p. However, in contrast to scerRap1p, scasRap1p was found to bind the human telomeric sequence. Moreover, the scasRap1p was found to incorporate a variant repeat in its binding to the otherwise homogeneous telomeric DNA of S. castellii. This ability to bind various sites differing in DNA sequence indicates a high degree of adjustability in the binding of scasRap1p to DNA.

Promoter-specific or all wRAPped up in the genome?

A question that has puzzled biologists is ‘what determines where, when and how regulatory proteins bind to the genome?’ These DNA-binding proteins control the expression of specific genes and are of fundamental importance for eukaryotic cell growth and survival. In Saccharomyces cerevisiae, repressor activator protein 1 (Rap1) is one such protein, which, together with the accessory silencing proteins Sir2, Sir3 and Sir4, functions to regulate telomere length and mating. Independent of Sir proteins, Rap1 is an essential sequence-specific transcriptional regulator of many crucial S. cerevisiae genes, acting either directly as a transcriptional activator or as an accessory for binding by other regulatory proteins. By examining the specificity of Rap1–DNA binding across the entire yeast genome, Lieb et al. 1 Lieb, J.D. et al. (2001) Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat. Genet. 10.1038/ng569 (http://www.nature.com/ng/) Google Scholar now provide some interesting answers to this fundamental biological question.

Promoter-specific or all wRAPped up in the genome?

A question that has puzzled biologists is ‘what determines where, when and how regulatory proteins bind to the genome?’ These DNA-binding proteins control the expression of specific genes and are of fundamental importance for eukaryotic cell growth and survival. In Saccharomyces cerevisiae, repressor activator protein 1 (Rap1) is one such protein, which, together with the accessory silencing proteins Sir2, Sir3 and Sir4, functions to regulate telomere length and mating. Independent of Sir proteins, Rap1 is an essential sequence-specific transcriptional regulator of many crucial S. cerevisiae genes, acting either directly as a transcriptional activator or as an accessory for binding by other regulatory proteins. By examining the specificity of Rap1–DNA binding across the entire yeast genome, Lieb et al. 1xSee all References1 now provide some interesting answers to this fundamental biological question.Yeast chromatin fragments enriched in stabilized Rap1 and Sir protein complexes were hybridized to DNA microarrays containing 12 943 DNA segments that represented every coding and intergenic region of the yeast genome. This novel approach enabled Lieb and colleagues to construct a genome-wide map of DNA–protein interactions. Careful analysis of the data identified the specific genomic targets of Rap1 and the Sir proteins. Consistent with the ability of these regulatory proteins to bind to telomeres, telomeric DNA was enriched in the Rap1 protein complexes.The analysis also allowed the identification of genes that Rap1 might regulate. To their delight, Lieb et al. found that Rap1 bound to the intergenic region immediately upstream of 362 genes. The protein products of these genes include ribosomal proteins, a continuous enzymatic pathway in glycolysis and others involved in protein synthesis. However, the most exciting finding was that, although the binding sequence for Rap1 occurs throughout the genome, Rap1 binds specifically to intergenic sequences containing potential promoters, rather than to coding sequences. This result raises the possibility that such a phenomenon might be a general property of proteins that function as transcriptional regulators but recognize DNA motifs found throughout the genome. There is no question that such a finding suggests the existence of a genome-wide mechanism that distinguishes between the potential DNA-binding sites that occur within coding regions and those in intergenic regions. The race is now on to understand how these promoter regions are ‘marked’. In the meantime, we eagerly await the results of the next study to use this powerful genomic mapping approach to identify the in vivo binding sites of other transcription factors with unknown DNA-binding specificities.

Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association.

We determined the distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions.

Alternative Mechanisms of Transcriptional Activation by Rap1p

Single Rap1p DNA-binding sites are poor activators of transcription of yeast minimal promoters, even when fully occupied in vivo. This low efficiency is due to two independent repression mechanisms as follows: one that requires the presence of histones, and one that requires Hrs1p, a component of the RNA polymerase II mediator complex. Both repression mechanisms were greatly reduced for constructs with tandemly arranged sites. In these constructs, UASrpg sequences (ACACCCATACATTT) activated better than telomere-like sequences (ACACCCACACACCC) in an orientation-dependent manner. Both mutations in the SWI/SNF complex and a deletion of amino acids 597--629 of Rap1p (Tox domain) decreased synergistic effects of contiguous telomeric sites. Conversely, deletion of amino acids 700--798 of Rap1p (Sil domain) made UASrpg and telomeric sites functionally indistinguishable. We propose that the Sil domain masks the main transactivation domain of Rap1p in Rap1p-telomere complexes, where the Tox domain behaves as a secondary activation domain, probably by interacting with chromatin-remodeling complexes. Rap1p DNA-binding sites in ribosomal protein gene promoters are mainly UASrpg-like; their replacement by telomeric sequences in one of these promoters (RPS17B) decreased transcription by two-thirds. The functional differences between UASrpgs and telomeric sequences may thus contribute to the differential expression of Rap1p-regulated promoters in vivo.

The N-terminal and C-terminal domains of RAP1 are dispensable for chromatin opening and GCN4-mediated HIS4 activation in budding yeast.

Repressor activator protein 1 (RAP1) assists GCN4-mediated HIS4 activation by overcoming some repressive aspect of chromatin structure to facilitate GCN4 binding. RAP1 also participates in other nuclear processes, and discrete domains of RAP1 have been shown to have specific properties including DNA binding, DNA bending, transcriptional activation, and silencing and telomere functions. To investigate whether specific domains of RAP1 are required to "open" chromatin and help GCN4 to activate the HIS4 gene, we examined the abilities of different truncated RAP1 proteins to perturb positioned nucleosomes via a nucleosomal RAP1 site in a yeast episome in vivo, and we tested HIS4 activation in yeast strains harboring truncated RAP1 mutants. We found that neither the DNA bending domain nor the putative activation domain of RAP1 is required for its ability to perturb the chromatin structure of a plasmid containing a RAP1 site. Similarly, neither the putative activation domain nor the N-terminal DNA-bending domain was required for GCN4-mediated activation of HIS4. We also used a rap1(ts) mutant to show that continuous occupancy of the HIS4 promoter by RAP1 is required for GCN4-mediated gene activation.

Specific interactions of the telomeric protein rap1p with nucleosomal binding sites

The telomeres of Saccharomyces cerevisiae are structurally and functionally well characterized. Their telomeric DNA is packaged by the protein Rap1p (repressor activator protein 1). Rap1p is a multifunctional, sequence-specific, DNA-binding protein which, besides participating in the regulation of telomeres structure and length, is also involved in transcriptional regulation of genes essential for cell growth and in silencing. Whereas the long tracts of telomeric DNA repeats of higher eukaryotes are mostly organized in closely spaced canonical nucleosomal arrays, it has been proposed that the 300 base-pairs of S. cerevisiae telomeric DNA are organized in a large non-nucleosomal structure that has been called the telosome. Recently, nucleosomes have been found also in Tetrahymena thermophila telomeres, suggesting that, in general, telomere structural differences between lower and higher eukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a stable ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is to place a single Rap1p-binding site at different positions within a nucleosome core and then test the binding of Rap1p and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the location of the site within the nucleosome core structure. These results show that a ternary complex between a nucleosome and Rap1p is stable and could be a possible intermediate between telomeric nucleosomes and telosomes in the dynamics of S. cerevisiae telomere organization.

A carbon-source-responsive element is required for regulation of the hypoxic ADP/ATP carrier (AAC3) isoform in Saccharomyces cerevisiae

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three genes that are differentially expressed under different physiological conditions. We investigated the transcriptional control of AAC3, an oxygen-repressed isoform. By deletion analysis, DNA electrophoretic mobility-shift assays, DNase I footprinting and site-directed mutagenesis, we have identified a promoter region (upstream repressing sequence 1, URS1) involved in a carbon-source-dependent repression of AAC3. It is different from the previously characterized oxygen-dependent ROX1 (regulation by oxygen 1) repressor-binding region (URS2). The complex character of URS1 includes the presence of two different cis-acting sequences: (i) a RAP1 (repressor activator protein 1)-binding site that is capable of binding the RAP1 protein in vitro and (ii) two putative ethanol-repression sequences, the modification of which derepresses the AAC3 gene. These findings demonstrate that the hypoxic AAC3 gene is regulated by two upstream repressor sites; one controlled by oxygen and haem, the other by the carbon source. Both sites function to completely switch off the expression of the AAC3 isoform when ATP is made by oxidative phosphorylation, and they modulate AAC3 expression when import of glycolytic ATP into mitochondria is required.

A carbon-source-responsive element is required for regulation of the hypoxic ADP/ATP carrier (AAC3) isoform in Saccharomyces cerevisiae

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three genes that are differentially expressed under different physiological conditions. We investigated the transcriptional control of AAC3, an oxygen-repressed isoform. By deletion analysis, DNA electrophoretic mobility-shift assays, DNase I footprinting and site-directed mutagenesis, we have identified a promoter region (upstream repressing sequence 1, URS(1)) involved in a carbon-source-dependent repression of AAC3. It is different from the previously characterized oxygen-dependent ROX1 (regulation by oxygen 1) repressor-binding region (URS(2)). The complex character of URS(1) includes the presence of two different cis-acting sequences: (i) a RAP1 (repressor activator protein 1)-binding site that is capable of binding the RAP1 protein in vitro and (ii) two putative ethanol-repression sequences, the modification of which derepresses the AAC3 gene. These findings demonstrate that the hypoxic AAC3 gene is regulated by two upstream repressor sites; one controlled by oxygen and haem, the other by the carbon source. Both sites function to completely switch off the expression of the AAC3 isoform when ATP is made by oxidative phosphorylation, and they modulate AAC3 expression when import of glycolytic ATP into mitochondria is required.

A Functional Comparison of BRCA1 C-terminal Domains in Transcription Activation and Chromatin Remodeling

The BRCA1 C-terminal (BRCT) domain is present in a number of proteins that are involved in various aspects of chromosomal events. The BRCT domain of BRCA1 is important for its function in DNA repair and transcriptional activation. When tethered to chromosomal DNA, this region of BRCA1 is capable of inducing changes in chromatin structure. Despite the sequence homology and functional proximity shared by the BRCT-containing proteins, it is not clear whether different BRCT domains confer a common biochemical activity. Much less is known about the functional significance of the characteristic amino acid residues in the BRCT motif. Here, we show that chromatin remodeling and transcription activation is not a common feature of BRCT domains. However, the BRCT domain of the multi-functional repressor-activator protein 1 (RAP1) can activate transcription and remodel chromatin in a manner similar to that shown for the BRCA1 BRCT domain. Most of the conserved amino acid residues in the second BRCA1 BRCT domain are essential for its function in transcriptional activation. In contrast, mutations of many analogous amino acid residues in the RAP1 BRCT domain greatly elevate the transcriptional activity. These data indicate that the conserved residues in these two BRCT domains may play different roles in transcriptional activation.

Rap1p-binding Sites in the Saccharomyces cerevisiae GPD1 Promoter Are Involved in Its Response to NaCl

Mechanisms involved in transcriptional regulation of the osmotically controlled GPD1 gene in Saccharomyces cerevisiae were investigated by promoter analysis. The GPD1 gene encodes NAD(+)-dependent glycerol-3-phosphate dehydrogenase, a key enzyme in the production of the compatible solute glycerol. By analysis of promoter deletions, we identified a region at nucleotides -478 to -324, in relation to start of translation, to be of great importance for both basal activity and osmotic induction of GPD1. Electrophoretic mobility shift and DNase I footprint analyses demonstrated protein binding to parts of this region that contain three consensus sequences for Rap1p (repressor activator protein 1)-binding sites. Actual binding of Rap1p to this region was confirmed by demonstrating enhanced electrophoretic mobility of the protein-DNA complex with extracts containing an N-terminally truncated version of Rap1p. The detected Rap1p-DNA interactions were not affected by changes in the osmolarity of the growth medium. Specific inactivation of the Rap1p-binding sites by a C-to-A point mutation in the core of the consensus showed that this factor is a major determinant of GPD1 expression since mutations in all three putative binding sites for Rap1p strongly hampered osmotic induction and drastically lowered basal activity. We also show that the Rap1p-binding sites appear functionally distinct; the most distal site (core of the consensus at position -386) exhibited the highest affinity for Rap1p and was strictly required for low salt induction (< or =0.6 m NaCl), but not for the response at higher salinities (> or =0.8 m NaCl). This indicates tha different molecular mechanisms might be operational for low and high salt responses of the GPD1 promoter.

RAP, RAP, open up! New wrinkles for RAP1 in yeast

RAP1 (repressor/activator protein 1) from budding yeast is well known for its involvement in gene activation and repression, telomere structure and function, and replication. Recent studies have examined additional roles for RAP1 in heterochromatin boundary-element formation, creation of hotspots for meiotic recombination, and chromatin opening. These studies provide new insight into the ability of this abundant DNA-binding protein to participate in a diverse array of functions taking place in a chromatin environment.

Cra-dependent transcriptional activation of the icd gene of Escherichia coli.

The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1.

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p.DNA. Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p.DNA.Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.

Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex.

Regulation of expression of the ethanol dehydrogenase gene (adhE) in Escherichia coli by catabolite repressor activator protein Cra.

The adhE gene encodes ethanol dehydrogenase and is located at min 27.9 of the Escherichia coli chromosome. Expression of adhE is about 10-fold higher in cells grown anaerobically than in cells grown aerobically and is dependent on both transcriptional and posttranscriptional factors. In this study, a trans-regulatory element repressing adhE expression was characterized by genetic and biochemical approaches. A mutation downregulating adhE expression was mapped at min 2 of the chromosome. DNA sequence analysis revealed a missense mutation in the cra gene, formerly known as fruR. The cra gene encodes a catabolite repressor-activator protein (Cra) involved in the modulation of carbon flow in E. coli. The mutant protein (Cra*) sustained an Arg148-->His substitution causing 1.5- and 3-fold stronger repression of adhE transcription under anaerobic and aerobic conditions, respectively. By contrast, cra null mutants displayed 1.5- and 4-fold increased adhE transcription under those conditions. Disruption of the cra gene did not abolish the anaerobic activation of the adhE gene but diminished it twofold. Cra and Cra* were purified as fusion proteins tagged with an N-terminal 6xHis element. In vitro, both fusion proteins showed binding to the adhE promoter region and to the control fruB promoter region, which is a known Cra target. However, only 6xHis-tagged Cra, and not 6xHis-Cra*, was displaced from the DNA target by the effector, fructose-1-phosphate (F1P), suggesting that the mutant protein is locked in a promoter-binding conformation and is no longer responsive to F1P. We suggest that Cra helps to tighten the control of adhE transcription under aerobic conditions by its repression.

Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein.

The Escherichia coli nir and nrf operons, which encode alternative nitrite reductases expressed during anaerobic growth, are subject to catabolite regulation. Transcription from the nir promoter is maximal when bacteria are grown in rich media such as Lennox broth supplemented with glucose. Conversely, expression of the nrf operon is suppressed by rich media, but stimulated during growth in minimal medium with glycerol and fumarate. The role of the catabolite repressor-activator (Cra) protein in catabolite regulation of the nir and nrf promoters was investigated. Transcription from the nir promoter was repressed by Cra when cells were grown in minimal medium with glycerol and fumarate. Crude protein extracts from a strain overproducing Cra encoded on a multicopy plasmid retarded a nir promoter fragment in a mobility shift assay, confirming that the observed Cra-dependent repression was due to the direct interaction of Cra with the regulatory region of the nir operon. Furthermore, the inclusion of fructose 1-phosphate, an effector of Cra DNA-binding activity, in the assay decreased the ability of Cra to retard the nir promoter fragment. In contrast, transcription from the nrf promoter was not regulated by Cra under any of the growth conditions tested.

A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans.

Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.

Cooperative interaction between Cra and Fnr in the regulation of the cydAB operon of Escherichia coli.

In vivo and in vitro experiments are reported demonstrating that the catabolite repressor-activator (Cra) protein (formerly designated FruR) regulates expression of the cydAB operon of Escherichia coli encoding cytochrome d oxidase. The Fnr protein is required for Cra-mediated transcriptional control, but the ArcA protein antagonizes the response to Cra. The results establish that Fnr, ArcA, and Cra exert their effects in an interdependent fashion.

Cra and the control of carbon flux via metabolic pathways

The catabolite repressor-activator (Cra) protein controls the direction of carbon flux through metabolic pathways in enteric bacteria. Cra binds to the control regions of target genes and exerts a negative effect on the expression of genes encoding glycolytic and Entner-Duodoroff enzymes, while exerting a positive effect on genes encoding Krebs cycle, glyoxylate shunt and gluconeogenic enzymes. Cra mediates cylic AMP-independent catabolite repression of positively Cra-regulated genes and catabolite activation of negatively Cra-controlled genes.