Ribosomal Gene Promoter Research Articles

Characterization of a fission yeast subunit of an RNA polymerase I essential transcription initiation factor, SpRrn7h/TAFI68, that bridges yeast and mammals: association with SpRrn11h and the core ribosomal RNA gene promoter

Production of eukaryotic ribosomal RNAs (rRNAs) entails sequence-specific recognition of regulatory sequences in the rRNA gene promoter. A putative subunit of the Schizosaccharomycespombe essential transcription initiation factor for rRNA synthesis has been identified that shares homology with both murine TAFI68 and Saccharomycescerevisiae Rrn7p, subunits of their species' transcription initiation factor. Affinity purified putative SpRrn7h and associated factors, including a putative Rrn11p homolog, SpRrn11h, bear RNA polymerase I transcription initiation factor activity, and recombinant SpRrn7h associates with S.pombe core rDNA promoter sequences. In the first widespread search for putative homologs of SpRrn7h/murine TAFI68, and SpRrn11h/murine TAFI48, multiple ones were identified across eukaryotes. Analysis of residues conserved between the fission yeast and murine essential initiation factor subunits aided in these identifications. Sequences in the core rRNA gene promoter contributing to transcriptional activation were investigated, including a perfect TATAAA element located at −35.

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.

A step subsequent to preinitiation complex assembly at the ribosomal RNA gene promoter is rate limiting for human RNA polymerase I-dependent transcription.

The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contained the factors required to support initiation of transcription, SL1, upstream binding factor (UBF), and Pol I. We have demonstrated that, throughout a single round of transcription, SL1 and UBF remain promoter bound. Moreover, the promoter-bound SL1 and UBF retain the ability to function in transcription initiation. SL1 has a central role in the stable association of the PIC with the promoter DNA. The polymerase component of the PIC is released from the promoter during transcription yet is efficiently recycled and able to reinitiate from "poised" promoters carrying SL1 and UBF, since the PICs captured on the immobilized templates sustained multiple rounds of transcription. Kinetic analyses of initiation of transcription by Pol I revealed that Pol I-dependent transcription is rate limited in a step subsequent to recruitment and assembly of Pol I PICs. The rate of RNA synthesis is primarily determined by the rates at which the polymerase initiates transcription and escapes the promoter, referred to as promoter clearance. This rate-limiting step in Pol I transcription is likely to be a major target in the regulation of rRNA gene expression.

Modeling of DNA local parameters predicts encrypted architectural motifs in Xenopus laevis ribosomal gene promoter.

We have modeled local DNA sequence parameters to search for DNA architectural motifs involved in transcription regulation and promotion within the Xenopus laevis ribosomal gene promoter and the intergenic spacer (IGS) sequences. The IGS was found to be shaped into distinct topological domains. First, intrinsic bends split the IGS into domains of common but different helical features. Local parameters at inter-domain junctions exhibit a high variability with respect to intrinsic curvature, bendability and thermal stability. Secondly, the repeated sequence blocks of the IGS exhibit right-handed supercoiled structures which could be related to their enhancer properties. Thirdly, the gene promoter presents both inherent curvature and minor groove narrowing which may be viewed as motifs of a structural code for protein recognition and binding. Such pre-existing deformations could simply be remodeled during the binding of the transcription complex. Alternatively, these deformations could pre-shape the promoter in such a way that further remodeling is facilitated. Mutations shown to abolish promoter curvature as well as intrinsic minor groove narrowing, in a variant which maintained full transcriptional activity, bring circumstantial evidence for structurally-preorganized motifs in relation to transcription regulation and promotion. Using well documented X. laevis rDNA regulatory sequences we showed that computer modeling may be of invaluable assistance in assessing encrypted architectural motifs. The evidence of these DNA topological motifs with respect to the concept of structural code is discussed.

TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

Structure of the Human Ubiquitin Fusion Gene Uba80 (RPS27a) and One of Its Pseudogenes

Ubiquitin is a highly conserved 76 amino acid protein that is generated in the cell by proteolysis of larger proteins containing either polyubiquitin chains or ubiquitin fused to carboxyl extension proteins (CEPs). In humans, the two human ubiquitin-CEP genes are Uba80 and Uba52, which code for ubiquitin fused to ribosomal protein S27a and L40, respectively. Working from a recently generated physical map of human chromosome 2p16, we determined the genetic and physical location and the genomic structure of the Uba80 gene in its entirety. A comparison of Uba80 to Uba52 revealed that the two genes share a conserved 5′-end structure, but that the structure of the ubiquitin coding regions was not conserved. Analysis of 400 bp of the promoter of Uba80 revealed strong similarity not only to the Uba52 promoter, but also to the other known human ribosomal gene promoters that have been identified to date. Homology searches also detected the presence of a pseudogene for Uba80, and the structure of this sequence feature is also reported.

Fission yeast contains an rDNA binding activity that interacts specifically with regulatory sequences for ribosomal RNA synthesis

Basal level transcriptional initiation of fission yeast ribosomal RNA genes is dependent on the core ribosomal RNA gene promoter and is stimulated by an upstream rDNA promoter element and by regulatory sequences located in its ∼3.5 kb intergenic rDNA spacer. A Schizosaccharomyces pombe sequence-specific rDNA binding activity was characterized that interacted with the upstream rDNA promoter region and that associated with required RNA polymerase I transcription components in initial fractionation steps. The rDNA binding activity was further purified and found to specifically associate with a region of the rDNA promoter between −80 and −56. The promoter region required for stable binding correlates with that mediating activated levels of transcriptional initiation. This rDNA binding activity stimulates in vitro rRNA synthesis supported by templates bearing this upstream promoter domain but not by templates lacking it.

Expression of foreign proteins in Trypanosoma congolense

An expression vector was constructed to express foreign genes in Trypanosoma congolense. The foreign gene and a neomycin phophotransferase (NPT) gene are flanked by glutamate and alanine rich protein (GARP) gene processing signals and their expression is driven by a ribosomal RNA gene promoter. The plasmid is not maintained as an episome in T. congolense, but the NPT gene permits selection of cells in which the plasmid has integrated into the genome. We used this plasmid to express luciferase, green fluorescent protein and a surface protein of Trypanosoma brucei, glycine–proline–glutamate–glutamate–threonine procyclic acidic repetitive protein (GPEET PARP). The plasmid-derived GPEET PARP is expressed on the surface of procyclic T. congolense and comigrates on a polyacrylamide gel with native GPEET PARP from T. brucei procyclic cells. We also attempted to use the plasmid to overexpress a previously identified T. congolense cysteine protease. The plasmid-derived cysteine protease mRNA species occurs in the transfected cells, but we were unable to detect increased levels of protein or protease activity.

Carbon Regulation of Ribosomal Genes in Neurospora crassa Occurs by a Mechanism Which Does Not Require Cre-1, the Homologue of the Aspergillus Carbon Catabolite Repressor, CreA

Transcription of the ribosomal protein and 40S rRNA genes is coordinately regulated during steady state growth and carbon shifts in Neurospora crassa. Recognition sequences for the Aspergillus nidulans carbon catabolite repressor, CreA, overlap transcriptional elements of a 40S rRNA gene and the crp-2 ribosomal protein gene. They also occur in similar locations in the promoters of several other ribosomal protein genes. Substitutions encompassing the −74 and −167 CreA consensus sequences in the crp-2 promoter result in a decrease in transcription. A cDNA encoding the N. crassa homologue of CreA was cloned and designated Cre-1. The Cre-1 protein is 45% identical to CreA from A. nidulans. Cre-1 protein produced in Escherichia coli binds to the CreA sites in the promoters of the 40S rRNA and crp-2 genes. An amino acid change from histidine (92) to threonine changed the Cre-1 binding specificity from (5′G/CC/TGGG/AG3′) to (5′G/CC/TGGCG3′). Base substitutions in the Cre-1 binding sites of the crp-2 promoter disrupted binding of wildtype Cre-1 in vitro but had no effect on transcription during steady state growth or carbon shifts, indicating that regulation of ribosomal genes by carbon source is not mediated by Cre-1, but via different proteins binding the Cre-1 sites and the Dde boxes.

DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences.

High-level transcriptional activation of most ribosomal protein (rp) genes in Saccharomyces cerevisiae is promoted by the global DNA-binding factor Rap1p. The creation of the complete database of yeast rp gene promoter sequences enabled us to develop a computational selection strategy aimed at acquiring detailed information concerning the DNA-binding specificity of Rap1p. Rap1p sites in rp gene promoters are often found in duplicate, exhibiting strong preferences in both spacing and orientation. Using these preferences, a weight matrix was selected that represents the in vivo binding requirements of Rap1p. The resulting matrix renders the identification of functional Rap1p binding sites more accurate and allowed us to re-evaluate previous in vitro data. Tandemly arranged Rap1p binding sites appear to be typical for rp gene promoters and differ in preferred spacing from sites occurring in (sub)telomeric repeats. The preferred spacing that is found in duplicate Rap1p binding sites of rp gene promoters is restricted to a small window between 15 and 26 bp. This is proposed to reflect the borders within which binding co-operativity operates. The data presented clearly illustrate that computational selection strategies provide information that reaches beyond experimental data. The rp database is available at the url: http://www. chem.vu.nl/BMB/Database.html.

Transactivation of a ribosomal gene by simian virus 40 large-T antigen requires at least three activities of the protein.

Simian virus 40 large-T antigen transactivates the ribosomal genes which are transcribed by RNA polymerase (pol I), as well as genes that are dependent on either pol II or pol III. This report identifies regions and activities of T antigen that are required to transactivate a pol I-dependent rat ribosomal gene promoter. By using the rat ribosomal gene (rDNA) promoter linked to a chloramphenicol acetyltransferase gene, we show that at least three separable T-antigen regions are necessary to achieve wild-type levels of transactivation of rDNA in transiently transfected monkey cells. One activity depends on the region of T antigen shared with small-t antigen (T/t common region). A second activity maps to amino acids 109 to 626 and is highly sensitive to mutational inactivation. Complementation analyses suggest that at least one activity in this region is independent of and must be in cis with the activity within the T/t common region. In addition, a functional nuclear localization signal is required for maximal T-antigen-mediated transactivation of rat rDNA. The three activities work in concert to override cellular species-specific controls and transactivate the rat ribosomal gene promoter. Finally, we provide evidence that although the tumor suppressor protein Rb has been shown to repress a pol I-dependent promoter, transactivation of the rat rDNA promoter does not depend on T antigen's ability to bind the tumor suppressor product Rb.

Dimerization and HMG box domains 1-3 present in Xenopus UBF are sufficient for its role in transcriptional enhancement.

Transcription of Xenopus ribosomal genes by RNA polymerase I is directed by a stable transcription complex that forms on the gene promoter. This complex is comprised of the HMG box factor UBF and the TBP-containing complex Rib1. Repeated sequence elements found upstream of the ribosomal gene promoter act as RNA polymerase I-specific trans-criptional enhancers. These enhancers function by increasing the probability of a stable transcription complex forming on the adjacent promoter. UBF is required for enhancer function. This role in enhancement is distinct from that at the promoter and does not involve translocation of UBF from enhancer repeats to the promoter. Here we utilize an in vitro system to demonstrate that a combination of the dimerization domain of UBF and HMG boxes 1-3 are sufficient to specify its role in enhancement. We also demonstrate that the acidic C-terminus of UBF is primarilyresponsible for its observed interaction with Rib1. Thus, we have uncoupled the Rib1 interaction and enhancer functions of UBF and can conclude that direct interaction with Rib1 is not a prerequisite for the enhancer function of UBF.

A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription.

Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system. Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p. Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells. Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I-Rrn3p complex and were inactive in promoter-dependent transcription. Activity was restored by adding purified pol I-Rrn3p complex to extracts from stationary phase cells. The pol I-Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation. We propose that the formation and disruption of the pol I-Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation.

Cytosine methylation and nucleolar dominance in cereal hybrids.

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive.

RNA polymerase I from S. cerevisiae depends on an additional factor to release terminated transcripts from the template

Terminated transcripts were generated at the ends of linearized DNA templates and at DNA-bound lac repressor by in vitro transcription with highly enriched or purified yeast RNA polymerase I (pol I). The release of the synthesized transcripts from the DNA was analyzed using immobilized DNA as template for the transcription reaction. An additional activity distinguishable from pol I was necessary to remove the terminated RNA from the template. Efficiency of transcript release could be improved if a thymidine-rich DNA fragment was located upstream of the transcriptional arrest caused by the DNA-bound lac repressor. The release activity interacted with different forms of polymerases, pol I able to initiate on the ribosomal gene promoter and pol I only active in non-specific transcription.

RNA polymerase I transcription on nucleosomal templates: the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression.

Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator.

The Xenopus RNA polymerase I transcription factor, UBF, has a role in transcriptional enhancement distinct from that at the promoter.

Repeated sequence elements found upstream of the ribosomal gene promoter in Xenopus function as RNA polymerase I-specific transcriptional enhancers. Here we describe an in vitro system in which these enhancers function in many respects as in vivo. The principal requirement for enhancer function in vitro is the presence of a high concentration of upstream binding factor (UBF). This system is utilized to demonstrate that enhancers function by increasing the probability of a stable transcription complex forming on the adjacent promoter. Species differences in UBF are utilized to demonstrate that enhancers do not act by recruiting UBF to the promoter, rather UBF performs its own distinct role at the enhancers. UBF function in enhancement differs from that at the promoter, as it is flexible with respect to both the species of UBF and the enhancer element employed. Additionally, we identify a potential role for the mammalian UBF splice variant, UBF2, in enhancer function. We demonstrate that the TATA box binding protein (TBP)-containing component of Xenopus RNA polymerase I transcription, Rib1, can interact with an enhancer-UBF complex. This suggests a model in which enhancers act by recruiting Rib1 to the promoter.

Resolution of RNA polymerase I into dimers and monomers and their function in transcription.

We have further analyzed the requirements of yeast RNA polymerase I (pol I) to initiate transcription at the ribosomal gene promoter. Resolution of yeast whole cell extracts through several chromatographic steps yielded three protein fractions required for accurate initiation. One fraction is composed of TBP associated within a 240 kDa protein complex. The fraction contributing the RNA polymerase I (pol I) activity consists of dimeric and monomeric pol I under conditions optimal for in vitro transcription. The capability to utilize the ribosomal gene promoter correlates with monomeric pol I complexes which are possibly associated with further transcription factors. These initiation competent pol I complexes appeared to be resistant to high salt concentrations. Pol I dimers which represent the majority of the isolated pol I, can be reversibly dissociated into monomers and are only active in non-specific RNA synthesis, if single stranded DNA serves as a template. We suggest a model in which dimeric inactive pol I is converted into an active monomeric form that might be associated with other transcription factors to maintain a stable initiation competent complex.

Protein and DNA Requirements for the Transcription Factor IIIA-induced Distortion of the 5 S rRNA Gene Promoter

Transcription factor-induced DNA distortion has become a common theme in eukaryotic gene regulation. A number of techniques have been applied to the study of transcription factor-induced DNA bending and flexibility including electron microscopy, circular permutation gel analysis, helical phasing gel analysis and cyclisation kinetics in solution. We have applied these techniques in order to assess the role that specific DNA sequences and protein domains of transcription factor IIIA (TFIIIA) play in the TFIIIA-induced distortion of the Xenopus5 S ribosomal RNA gene promoter. Electron spectroscopic imaging analysis of TFIIIA : DNA com- plexes indicate that TFIIIA binding involves compaction of the 5 S promoter into a precise three-dimensional hairpin-shaped structure. This compaction can be detected utilising circular permutation gel analysis and the distortion results in an apparent bend angle of 55 to 60° near the centre of the TFIIIA binding site. Helical phasing analysis demonstrates that the 60° bend angle as measured by circular permutation can be detected as a static bend directed towards the minor groove between bases +63 and +64 of the 5 S rRNA gene. The amplitude of the TFIIIA : 5 S gene phasing signal is similar to the phasing signal obtained utilising bacterial CAP : DNA complexes with bend angles of approximately 90°. These results are supported by phased ligase-mediated cyclisation kinetics in solution. Analysis of DNA deletion constructs indicate that the 5′ A block of the internal 5 S gene promoter, which is required for transcriptional activity, is also required for TFIIIA-induced distortion of the 5 S gene promoter. Analysis of the N-terminal papain fragment of TFIIIA indicates that the 34 kDa zinc finger DNA binding domain is sufficient for compaction of the 5 S gene promoter. These results are discussed in relation to the modular model of TFIIIA : DNA interaction in which individual zinc fingers contribute to the protein-induced distortion of the DNA helix and overall DNA binding affinity in a complex, non-additive fashion.

Upstream binding factor stabilizes Rib 1, the TATA-binding-protein-containing Xenopus laevis RNA polymerase I transcription factor, by multiple protein interactions in a DNA-independent manner.

Initiation of RNA polymerase I transcription in Xenopus laevis requires Rib 1 and upstream binding factor (UBF). UBF and Rib 1 combine to form a stable transcription complex on the Xenopus ribosomal gene promoter. Here we show that Rib 1 comprises TATA-binding protein (TBP) and TBP-associated factor components. Thus, Rib 1 is the Xenopus equivalent of mammalian SL 1. In contrast to SL 1, Rib 1 is an unstable complex that readily dissociates into TBP and associated components. We identify a novel function for UBF in stabilizing Rib 1 by multiple protein interactions. This stabilization occurs in solution in a DNA-independent manner. These results may partially explain the difference in UBF requirement between Xenopus and mammalian systems.