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PubMed2001

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5'-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the 'AU-box', just upstream of the SD sequences. To clarify the role of 5'-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 -38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the -38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5'-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5'-UTR and possible roles of the AU-box motif and the SD sequence.

5' Untranslated Regions↗

PubMed1999

Human subtilisin-like proprotein convertase, PACE4 (SPC4) gene expression is highly regulated through E-box elements in HepG2 and GH4C1 cells.

PACE4 (SPC4) is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which participates in maturation of precursor proteins. PACE4 is expressed at high levels in the anterior pituitary, central nervous system, the developing olfactory bulb, heart, and liver. Recently, we determined the gene structure of human PACE4. [Tsuji et al. (1997) J. Biochem. 122, 438-452]. The 5'-flanking region of PACE4 gene contains 12 E-boxes (E1 to E12) within 1 kb upstream of the transcription initiation site. To examine the function of these E-box elements in the regulation of PACE4 expression, deletion and mutation constructs of the 5'-flanking region were ligated to the luciferase gene and analyzed for promoter activity in HepG2 and GH4C1 cells, which express PACE4 at high level. Some differences were observed in the activity of each promoter construct between HepG2 and GH4C1 cells, although the overall profiles of activity for the promoter fragment series were similar regardless of cell type. We showed that the basal promoter activity of the PACE4 gene is first determined by sequences lying between -315 and -1 bp and further regulated by positive and negative elements in the upstream region. Site-directed mutagenesis of E-boxes in these regulatory elements showed that the E10 E-box act as positive regulator, whereas an E-box cluster (E4-E9) acts as a negative regulator in both cells. E2 E-box acts as a positive regulator only in HepG2 cells. Other E-boxes (E1, E3, and E12) had no effect on the promoter activity. These results indicate that E-box elements play a critical role in controlling PACE4 expression in HepG2 and GH4C1 cells and that PACE4 expression is regulated by a mechanism distinct from that of other SPC family proteases.

Base Sequence↗

PubMed2002

Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit.

The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. Here, we investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. We find that addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/C(Cdh1). The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C.

3T3 Cells↗

PubMed2003

A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis.

The PDF1.2 gene of Arabidopsis encoding a plant defensin is commonly used as a marker for characterization of the jasmonate-dependent defense responses. Here, using PDF1.2 promoter-deletion lines linked to the beta-glucoronidase-reporter gene, we examined putative promoter elements associated with jasmonate-responsive expression of this gene. Using stably transformed plants, we first characterized the extended promoter region that positively regulates basal expression from the PDF1.2 promoter. Second, using promoter deletion constructs including one from which the GCC-box region was deleted, we observed a substantially lower response to jasmonate than lines carrying this motif. In addition, point mutations introduced into the core GCC-box sequence substantially reduced jasmonate responsiveness, whereas addition of a 20-nucleotide-long promoter element carrying the core GCC-box and flanking nucleotides provided jasmonate responsiveness to a 35S minimal promoter. Taken together, these results indicated that the GCC-box plays a key role in conferring jasmonate responsiveness to the PDF1.2 promoter. However, deletion or specific mutations introduced into the core GCC-box did not completely abolish the jasmonate responsiveness of the promoter, suggesting that the other promoter elements lying downstream from the GCC-box region may also contribute to jasmonate responsiveness. In other experiments, we identified a jasmonate- and pathogen-responsive ethylene response factor transcription factor, AtERF2, which when overexpressed in transgenic Arabidopsis plants activated transcription from the PDF1.2, Thi2.1, and PR4 (basic chitinase) genes, all of which contain a GCC-box sequence in their promoters. Our results suggest that in addition to their roles in regulating ethylene-mediated gene expression, ethylene response factors also appear to play important roles in regulating jasmonate-responsive gene expression, possibly via interaction with the GCC-box.

Arabidopsis↗

PubMed2005

A tertiary structural element in S box leader RNAs is required for S-adenosylmethionine-directed transcription termination.

The S box transcription termination control system regulates expression of genes involved in methionine metabolism. Expression of the S box regulon, comprised of 11 transcriptional units in Bacillus subtilis, is induced in response to starvation for methionine. We previously demonstrated that S-adenosylmethionine (SAM) is the molecular effector sensed by the S box leader RNAs during transcription. A secondary structure model for S box leader RNAs was developed based on conservation of primary sequence elements and sequence covariation in helical domains. Covariation of nucleotides in two distantly spaced unpaired regions in the S box leader RNAs suggested that these two domains might interact in the RNA tertiary structure. In this study, site-directed mutagenesis of the covarying residues in two B. subtilis S box leader sequences was employed to test the hypothesis that base-pairing between these regions may be important. The effect of these mutations on in vivo expression, transcription termination in vitro, SAM binding, and leader RNA structure strongly supported the model that interaction between these two regions plays a key role in S box leader function. This provides the first insight into the three-dimensional arrangement of structural elements within the S box RNAs.

5' Untranslated Regions↗

PubMed1995

The MADS-box family of transcription factors.

The MADS-box family of transcription factors has been defined on the basis of primary sequence similarity amongst numerous proteins from a diverse range of eukaryotic organisms including yeasts, plants, insects, amphibians and mammals. The MADS-box is a conserved motif found within the DNA-binding domains of these proteins and the name refers to four of the originally identified members: MCM1, AG, DEFA and SRF. Several proteins within this family have significant biological roles. For example, the human serum-response factor (SRF) is involved in co-ordinating transcription of the protooncogene c-fos, whilst MCM1 is central to the transcriptional control of cell-type specific genes and the pheromone response in the yeast Saccharomyces cerevisiae. The RSRF/MEF2 proteins comprise a sub-family of this class of transcription factors which are key components in muscle-specific gene regulation. Moreover, in plants, MADS-box proteins such as AG, DEFA and GLO play fundamental roles during flower development. The MADS-box is a contiguous conserved sequence of 56 amino acids, of which 9 are identical in all family members described so far. Several members have been shown to form dimers and consequently two functional regions within the MADS-box have been defined. The N-terminal half is the major determinant of DNA-binding specificity whilst the C-terminal half is necessary for dimerisation. This organisation allows the potential formation of numerous proteins, with subtly different DNA-binding specificities, from a limited number of genes by heterodimerisation between different MADS-box proteins. The majority of MADS-box proteins bind similar sites based on the consensus sequence CC(A/T)6GG although each protein apparently possesses a distinct binding specificity. Moreover, several MADS-box proteins specifically recruit other transcription factors into multi-component regulatory complexes. Such interactions with other proteins appears to be a common theme within this family and play a pivotal role in the regulation of target genes.

Amino Acid Sequence↗

PubMed1996

Specific binding of DnaA protein to a DnaA box in the guaB gene of Escherichia coli K12.

Expression of the guaBA operon of Escherichia coli is regulated by the DNA replication-initiating protein, DnaA. Two DnaA boxes, which are potential binding sites for DnaA, are present in the gua operon. One box (with 8/9 match to the DnaA box consensus sequence) is at the gua promoter; the other box, which has a consensus sequence, is on the non-transcribed strand within the guaB coding region approximately 200 bp downstream of the initiation codon. The binding in vitro of purified DnaA protein to these boxes was investigated by filter retention and gel retardation analysis, and by deoxyribonuclease I footprinting, using restriction fragments of gua operon DNA. DnaA protein was shown to bind specifically only to the fragment carrying the consensus sequence DnaA box, and to protect this box from deoxyribonuclease I. Transcription termination resulting from the binding of DnaA to this box within the guaB gene explains repression by DnaA of the gua operon in vivo.

Bacterial Proteins↗

PubMed1999

The presence of two DnaA-binding sequences is required for an efficient interaction of the Escherichia coli DnaA protein with each particular weak DnaA box region.

Using an electron microscopic method for visualizing interactions of the Escherichia coli DnaA protein with weak DnaA-binding DNA sequences, we found that DnaA binds effectively to two separated weak DnaA box regions located on the same DNA fragment. As expected, no DnaA-DNA interactions were detected when both DnaA box regions were mutagenized to the sequence totally incapable of binding DnaA. However, when only one of these two regions was mutagenized, the lack of interactions between DnaA and DNA was observed not only at the scrambled DnaA box but also at the second weak DnaA box region. These results indicate that for the efficient binding of DnaA to a weak DnaA box region, the presence of at least two such DNA sequences is necessary. Our finding also suggests that binding of DnaA protein to weak DnaA box sequences may be cooperative. In addition, we found that DnaA-mediated transcription termination in vivo requires two DnaA boxes, one of them is a weak one. It seems, therefore, that some mechanisms of regulation of transcription and DNA replication by DnaA, that involve interactions of DnaA with weak DnaA boxes, may be more complicated than initially proposed.

Bacterial Proteins↗

PubMed1990

A single DnaA box is sufficient for initiation from the P1 plasmid origin.

The P1 plasmid replication origin requires the host DnaA protein for function. Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary. Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene. We show that either set alone is active for origin function. One of the two origin boxes is relatively inactive. Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus. This single consensus box is functional when placed either to the left or right of the core origin sequences. The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.

Bacterial Proteins↗

PubMed1995

Transcription termination in the Escherichia coli dnaA gene is not mediated by the internal DnaA box.

DnaA protein is a DNA-binding protein which recognizes a 9-bp consensus sequence called the DnaA box. By binding to DnaA boxes, DnaA protein regulates initiation of chromosomal replication and transcription of several genes. The dnaA gene contains two DnaA boxes, one located in the regulatory region and one within the structural gene. In this paper, we explore the role of the internal DnaA box in dnaA expression because it has been proposed that the DnaA box-DnaA protein complex can block transcribing RNA polymerase. Firstly, we analyzed the degree of derepression of the dnaA gene, measured as beta-galactosidase activity of a dnaA-lacZ fusion inserted onto the bacterial chromosome, produced by an extra copy number of the dnaA DnaA boxes carried by multicopy plasmids. Secondly, we analyzed repression produced by elevated levels of DnaA protein on single-copy dnaA-lacZ fusions containing or not containing the internal DnaA box. Our results indicate that the internal DnaA box does not play a regulatory role in dnaA expression.

Bacterial Proteins↗

PubMed1999

DnaA boxes are important elements in setting the initiation mass of Escherichia coli.

The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.

Bacterial Proteins↗

PubMed2004

The spatial organization of the VirR boxes is critical for VirR-mediated expression of the perfringolysin O gene, pfoA, from Clostridium perfringens.

The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR. Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O. For this study, we introduced mutated VirR boxes into a C. perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation. Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the -35 region are shown to be critical for perfringolysin O production. Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system. The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity. In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation. Finally, we show that the C. perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins. We propose that these interactions are required for VirR-mediated transcriptional activation.

Bacterial Proteins↗

PubMed1988

Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes.

Rous sarcoma virus nucleocapsid protein p12 (NC) contains two conserved amino acid motifs, the Cys-His boxes, which constitute potential metal-binding domains. To try to understand the function of NC and of each of its Cys-His boxes during the viral life cycle, particularly in viral RNA packaging, we have used synthetic oligonucleotides to delete precisely either the proximal or the distal box, or both Cys-His boxes. The mutant DNAs were transfected into chicken embryo fibroblasts, and the virions produced in a transient assay were characterized biochemically for production of viral proteins and particles, RNA packaging, and infectivity. The results indicated the following. (i) The deletion of either the proximal or the distal box decreases the amount of viral RNA packaged in the particles and results in incomplete 70S dimer formation. (ii) The deletion of both boxes inhibits viral RNA packaging. (iii) The deletion of the proximal, but not the distal, box suppresses any detectable infectivity, while the deletion of the distal, but not the proximal, box lowers infectivity 100 to 200 times.

Animals↗

PubMed1996

The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation.

ICP27 is an essential herpes simplex virus type 1 nuclear regulatory protein that is required for efficient viral gene expression. Although the mechanism by which ICP27 regulates genes is unknown, a variety of evidence suggests that it functions posttranscriptionally, and recent studies indicate that it is an RNA-binding protein. Previously, we noted that a short arginine- and glycine-rich sequence in ICP27 (residues 138 to 152) is similar to an RGG box motif, a putative RNA-binding determinant found in a number of cellular proteins (W. Mears, V. Lam, and S. Rice, J. Virol. 69:935-947, 1995). In the present study, we have further investigated ICP27's association with RNA and examined the role of the RGG box in RNA binding. We find that ICP27 binds efficiently to RNA homopolymers composed of poly(G) and weakly to poly(U) RNA homopolymers. Poly(G) binding activity maps to the N-terminal 189 residues of ICP27 and requires the RGG box sequence. Using a northwestern blotting assay, we demonstrate that the RGG box alone (residues 140 to 152) can mediate RNA binding when attached to a heterologous protein. As many cellular RGG box proteins are methylated on arginine residues, we also investigated the in vivo methylation status of ICP27. Our results demonstrate that ICP27 is methylated in herpes simplex virus-infected cells. Methylation is dependent on the presence of the RGG box, suggesting that one or more arginine residues in the RGG box sequence are modified. These data demonstrate that ICP27 displays the characteristics of an RGG box-type RNA-binding protein.

Amino Acid Sequence↗

PubMed1998

Functional analysis of the CAAT box in the major late promoter of the subgroup C human adenoviruses.

Comparisons among sequences predicted to encode the major late promoter (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation suggests that the CAAT box plays an important role in the function of the MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, in terms both of quantitative levels of transcription and of specificity, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically. The CAAT5 mutation had no discernible phenotype by itself but when coupled with the previously described USF0 mutation, which disrupts binding of the upstream stimulating factor (USF) but is otherwise phenotypically silent, gave rise to virus with a severe replication deficiency. Nuclear run-on assays showed that transcription initiation at the mutant MLP was significantly reduced compared with that of the wild type or the virus containing CAAT5 alone. Replication of the double mutant was lower than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further lowered the binding of transcription factor CP1 (also called CBF, NF-Y). Replacement of the CAAT box by an ATF binding site or an OCT1 binding site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial restoration of the wild-type phenotype. The failure to restore the functional redundancy normally exhibited by the CAAT box and the proximal upstream activating element is consistent with the idea that in the adenovirus MLP the CAAT box is preferred over others as the distal transcriptional element.

Adenoviruses, Human↗

PubMed2000

Processing of intron-encoded box C/D small nucleolar RNAs lacking a 5',3'-terminal stem structure.

The C and D box-containing (box C/D) small nucleolar RNAs (snoRNAs) function in the nucleolytic processing and 2'-O-methylation of precursor rRNA. In vertebrates, most box C/D snoRNAs are processed from debranched pre-mRNA introns by exonucleolytic activities. Elements directing accurate snoRNA excision are located within the snoRNA itself; they comprise the conserved C and D boxes and an adjoining 5',3'-terminal stem. Although the terminal stem has been demonstrated to be essential for snoRNA accumulation, many snoRNAs lack a terminal helix. To identify the cis-acting elements supporting the accumulation of intron-encoded box C/D snoRNAs devoid of a terminal stem, we have investigated the in vivo processing of the human U46 snoRNA and an artificial snoRNA from the human beta-globin pre-mRNA. We demonstrate that internal and/or external stem structures located within the snoRNA or in the intronic flanking sequences support the accumulation of mammalian box C/D snoRNAs lacking a canonical terminal stem. In the intronic precursor RNA, transiently formed external and/or stable internal base-pairing interactions fold the C and D boxes together and therefore facilitate the binding of snoRNP proteins. Since the external intronic stems are degraded during snoRNA processing, we propose that the C and D boxes alone can provide metabolic stability for the mature snoRNA.

Animals↗

PubMed1999

E-box and cyclic adenosine monophosphate response elements are both required for follicle-stimulating hormone-induced transferrin promoter activation in Sertoli cells.

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight into the regulation of Sertoli cell differentiation. Analysis of the mouse transferrin (mTf) promoter reveals the presence of a number of conserved response elements that have previously been shown to regulate cell specific expression of the human transferrin (hTf) promoter. One of these elements is the human PRII region, which is a cAMP response element (CRE)-like element that is more than 80% conserved in the mTf promoter. The activation of the hTf promoter by FSH and cAMP in rat Sertoli cells has been shown to be mediated in part through the CRE-like PRII region and binding of the CRE binding protein (CREB). The present study investigates the role of PRII in the activation of mTf promoter by FSH and cAMP in rat Sertoli cells. Mutations in the PRII of the mTf promoter reduced FSH activation by only 50% and cAMP activation by more than 90%. In contrast, the mutant PRII mTf promoter construct was fully activated by a partially purified testicular paracrine activity PModS(S300). Gel shift experiments demonstrated that proteins that can bind a consensus CRE oligonucleotide also bind the PRII region of the mTf promoter. An immunoblot confirmed that CREB binds the PRII and promotes the gel shift observed. The hypothesis developed was that another cis-acting element in addition to the CRE-like PRII is also involved in FSH actions. A conserved response element in both the mTf and hTf promoters is the basic helix-loop-helix (bHLH) responsive E-box sequence. Both FSH and PModS (S300) activity were found to promote a mTf E-box gel shift that contained the E2A gene product the bHLH protein E47. Interestingly, mutations in the E-box of the mTf promoter completely abolished the PModS(S300) activation and partially (52%) inhibited the activation by FSH. In contrast, the mutant E-box mTf promoter construct was fully activated by cAMP. Finally a double mutation of both the PRII and the E-box completely abolished FSH activation of the mTf promoter. These results suggest that optimal activation of the mouse transferrin promoter by FSH requires both CREB binding to the CRE-like PRII region and bHLH binding to the E-box. Information is provided that indicates a number of Sertoli cell promoters contain a close association of E-box and CRE-like elements. Observations are discussed in regards to the potential interactions of the CRE and E-box response elements in mediating FSH actions in Sertoli cells.

Animals↗

PubMed2006

Analysis of the binding of the N-terminal conserved domain of yeast Cbf5p to a box H/ACA snoRNA.

During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5(Delta)p) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5(Delta)p on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5(Delta)p can bind to a single stem-loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5-RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5' end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3' end of the molecule, including the conserved ACA box.

Amino Acid Sequence↗