Promoter Sequence Elements Research Articles

Molecular characterization of genes involved in tolerance of cadmium in Triticum aestivum (L.) under Cd stress

The NRAMPs (natural resistance-associated macrophage proteins) are major transporters for the absorption and transport of metals like Pb, Zn, Mn, Fe, and Cd in plants. While NRAMP gene family members have been extensively studied as metal transporters in model and other plants, little information has been reported on their role in Triticum aestivum, particularly in response to Cd stress. Current study reported 13 NRAMP candidates in the genome of T. aestivum. Phylogenetic analysis divided these into three clades. Motif and gene structure study showed that members in the same clades shared the same location and pattern, which further supported the phylogenetic analysis. The analysis of cis-acting elements in promoter sequences of NRAMP genes in wheat identified stress-responsive transcription factor binding sites. Multiple sequence alignment identified the conservation of important residues. Based on RNA-seq and qRT-PCR analysis, Cd stress-responsive variations of TaNRAMP gene expression were reported. This study provides comprehensive data to understand the TaNRAMP gene family, its features, and its expression, which will be a helpful framework for functional research.

Genome-wide identification and molecular expression profile analysis of FHY3/FAR1 gene family in walnut (Juglans sigillata L.) development

BackgroundJuglans sigillata L. (walnut) has a high economic value for nuts and wood and has been widely grown and eaten around the world. Light plays an important role in regulating the development of the walnut embryo and promoting nucleolus enlargement, which is one of the factors affecting the yield and quality of walnut. However, little is known about the effect of light on the growth and quality of walnuts. Studies have shown that far red prolonged hypocotyl 3 (FHY3) and far red damaged response (FAR1) play important roles in plant growth, light response, and resistance. Therefore, FHY3/FAR1 genes were identified in walnuts on a genome-wide basis during their growth and development to reveal the potential regulation mechanisms involved in walnut kernel growth and development.ResultsIn the present study, a total of 61 FHY3/FAR1 gene family members in walnuts have been identified, ranging in length from 117 aa to 895 aa. These gene family members have FHY3 or FAR1 conserved domains, which are unevenly distributed on the 15 chromosomes (Chr) of the walnut (except for the Chr16). All 61 FHY3/FAR1 genes were divided into five subclasses (I, II, III, IV, and V) by phylogenetic tree analysis. The results indicated that FHY3/FAR1 genes in the same subclasses with similar structures might be involved in regulating the growth and development of walnut. The gene expression profiles were analyzed in different walnut kernel varieties (Q, T, and F). The result showed that some FHY3/FAR1 genes might be involved in the regulation of walnut kernel ripening and seed coat color formation. Seven genes (OF07056-RA, OF09665-RA, OF24282-RA, OF26012-RA, OF28029-RA, OF28030-RA, and OF08124-RA) were predicted to be associated with flavonoid biosynthetic gene regulation cis-acting elements in promoter sequences. RT-PCR was used to verify the expression levels of candidate genes during the development and color change of walnut kernels. In addition, light responsiveness and MeJA responsiveness are important promoter regulatory elements in the FHY3/FAR1 gene family, which are potentially involved in the light response, growth, and development of walnut plants.ConclusionThe results of this study provide a valuable reference for supplementing the genomic sequencing results of walnut, and pave the way for further research on the FHY3/FAR1 gene function of walnut.

RNA activation in ticks

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3′- untranslated region (3’-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3ʹ-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

Synergistic actions of three MYB transcription factors underpins the high accumulation of myricetin in Morella rubra.

Flavonols are health-promoting bioactive compounds important for human nutrition, health, and plant defense. The transcriptional regulation of kaempferol and quercetin biosynthesis has been studied extensively, while little is known about the regulatory mechanisms underlying myricetin biosynthesis, which has strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. In this study, the flavonol-specific MrMYB12 in Morella rubra preferred activating the promoter of flavonol synthase 2 (MrFLS2) (6.4-fold) rather than MrFLS1 (1.4-fold) and upregulated quercetin biosynthesis. Furthermore, two SG44 R2R3-MYB members, MrMYB5 and MrMYB5L, were identified by yeast one-hybrid library screening using the promoter of flavonoid 3',5'-hydroxylase (MrF3'5'H), and transcript levels of these R2R3-MYBs were correlated with accumulation of myricetin derivatives during leaf development. Dual-luciferase and electrophoretic mobility shift assays demonstrated that both MrMYB5 and MrMYB5L could bind directly to MYB recognition sequence elements in promoters of MrF3'5'H or MrFLS1 and activate their expression. Protein-protein interactions of MrMYB5 or MrMYB5L with MrbHLH2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. MrMYB5L-MrbHLH2 showed much higher synergistic activation of MrF3'5'H or MrFLS1 promoters than MrMYB5-MrbHLH2. Studies with Arabidopsis thaliana homologs AtMYB5 and AtTT8 indicated that similar synergistic regulatory effects occur with promoters of MrF3'5'H or MrFLS1. Transient overexpression of MrMYB5L-MrbHLH2 in Nicotiana benthamiana induced a higher accumulation of myricetin derivatives (57.70 μg g-1 FW) than MrMYB5-MrbHLH2 (7.43 μg g-1 FW) when MrMYB12 was coexpressed with them. This study reveals a novel transcriptional mechanism regulating myricetin biosynthesis with the potential use for future metabolic engineering of health-promoting flavonols.

Co-expression and Interaction of Pax6 with Genes and Proteins of Immunological Surveillance in the Brain of Mice.

The Pax6 binds to promoter sequence elements of genes involved in immunological surveillance and interacts with Iba1, p53, Ras-GAP, and Sparc in the brain of mice. The Pax6 also affects the expression pattern of genes involved in neurogenesis and neurodegeneration. However, the expression and association of Pax6 in the brain under immunologically challenged conditions are still elusive. Therefore, it has been intended to analyze the association of Pax6 in the immunity of the brain using the immune-challenged Dalton's lymphoma (DL) mice model. The expressions of Pax6, Iba1, and Tmem119 decreased, but expressions of Ifn-γ, Tnf-α, Bdnf, and Tgf-β increased in the brain of immune-challenged mice as compared to the control. The level of co-expression of Pax6 decreased in dual positive cells with Iba1, Tmem119, Sparc, p53, Bdnf, and Tgf-β in the brain of immune-challenged mice. Binding of Pax6 to multiple sites of the promoter sequences of Bdnf and Tgf-β indicates their Pax6-associated differential expression and association with immune responsive gene. The levels of binding of Pax6 to Tmem119, Iba1, Ifn-γ, and Tnf-α got altered during the immune-challenged state as compared to control. Results provide the first evidence of the association of Pax6 in brain-specific immunity.

Rational design of minimal synthetic promoters for plants.

Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

Bioinformatic Identification of Chinese Hamster Ovary (CHO) Cold-Shock Genes and Biological Evidence of their Cold-Inducible Promoters.

Lower cultivation temperature dramatically affects cell growth and cellular productivity in Chinese hamster ovary cells (CHO) and is often used in industrial applications with the aim to enhance productivity. Cold-inducible proteins whose activity is induced at lower temperature play an important role in understanding the mechanisms of cold-induced changes in gene expression. One of these mechanisms is increased transcription of specific target genes controlled by sequence elements in cold-inducible promoters. This study provides a list of cold-inducible genes and endogenous cold-inducible promoters of CHO cells. Transcriptome data before and after a temperature shift from 37 to 33°C are analyzed to identify 94 cold-inducible genes, which are highly expressed and have a high fold change in expression after the temperature shift. Cold-inducible promoters are identified from the top ten cold-inducible genes, showing up to 11-fold increased luciferase expression at lowered temperature in transient transfections. Additionally, several common transcription factor binding sites are identified in the top cold-inducible promoter sequences and expression of their corresponding transcription factors over temperature-shift cultivation is evaluated. These may be responsible for enhanced promoter activity under lower temperature and can be used as engineering targets.

Brassica rapa expansin-like B1 gene (BrEXLB1) regulate growth and development in transgenic Arabidopsis and elicits response to abiotic stresses

The functional role of expansin-like A and B family members remains unclear in plants. In this study, we investigate the functional role of Brassica rapa expansin-like B1 (BrEXLB1) by overexpressing BrEXLB1 sense, antisense and BrEXLB1 specific promoter: GUS in Arabidopsis. The overexpressors of sense BrEXLB1, antisense BrEXLB1 showed variable and unstable pleiotropic effects on leaf growth. Interestingly, overexpressors of sense BrEXLB1 enhances plant height than antisense overexpressors and wild types. GUS reporter-aided analysis of BrEXLB1 promoter showed their activity prominently in seeds, roots and carpels. This is further confirmed by relative quantification of BrEXLB1 among various tissue samples of B. rapa by semi-quantitative Reverse transcription-polymerase chain reaction assay (RT-PCR). We found that BrEXLB1 promoter has several cis-acting elements including light-responsive, phytohormone-responsive, environmental/stress-responsive and tissue-specific elements in promoter sequences. Hence, we analysed the promoter activity of BrEXLB1 by GUS reporter-aided approach, in which stress, phytohormones and other factors regulated changes in GUS transcripts were measured using quantitative RT-PCR. This study suggests that promoter activity is inducible to exogenous application of phytohormones such as indole-3-acetic acid, jasmonic acid, and other factors such as white light and drought stress. These results suggest that BrEXLB1 under its specific promoters may participate in regulation of leaf, plant growth and responds to hormone availability, light quality, dark periods, developmental stages, and drought conditions.

Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5′ mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

Endogenous Locus Reporter Assays.

Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

Prediction of bipartite transcriptional regulatory elements using transcriptome data of Arabidopsis.

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of ‘4 bases—a spacer with a flexible length—4 bases’. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein–DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.

LDSS-P: an advanced algorithm to extract functional short motifs associated with coordinated gene expression

Identifying functional elements in promoter sequences is a major goal in computational and experimental genome biology. Here, we describe an algorithm, Local Distribution of Short Sequences for Prokaryotes (LDSS-P), to identify conserved short motifs located at specific positions in the promoters of co-expressed prokaryotic genes. As a test case, we applied this algorithm to a symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti. The LDSS-P profiles that overlap with the 5′ section of the extracytoplasmic function RNA polymerase sigma factor RpoE2 consensus sequences displayed a sharp peak between -34 and -32 from TSS positions. The corresponding genes overlap significantly with RpoE2 targets identified from previous experiments. We further identified several groups of genes that are co-regulated with characterized marker genes. Our data indicate that in S. meliloti, and possibly in other Rhizobiaceae species, the master cell cycle regulator CtrA may recognize an expanded motif (AACCAT), which is positionally shifted from the previously reported CtrA consensus sequence in Caulobacter crescentus. Bacterial one-hybrid experiments showed that base substitution in the expanded motif either increase or decrease the binding by CtrA. These results show the effectiveness of LDSS-P as a method to delineate functional promoter elements.

Taspase1 processing alters TFIIA cofactor properties in the regulation of TFIID

TFIIA is an important positive regulator of TFIID, the primary promoter recognition factor of the basal RNA polymerase II transcription machinery. TFIIA antagonises negative TFIID regulators such as negative cofactor 2 (NC2), promotes specific binding of the TBP subunit of TFIID to TATA core promoter sequence elements and stimulates the interaction of TBP-associated factors (TAFs) in the TFIID complex with core promoter elements located downstream of TATA, such as the initiator element (INR). Metazoan TFIIA consists of 3 subunits, TFIIAα (35 kDa), β (19 kDa) and γ (12 kDa). TFIIAα and β subunits are encoded by a single gene and result from site-specific cleavage of a 55 kDa TFIIA(α/β) precursor protein by the protease Taspase1. Metazoan cells have been shown to contain variable amounts of TFIIA (55/12 kDa) and Taspase1-processed TFIIA (35/19/12 kDa) depending on cell type, suggesting distinct gene-specific roles of unprocessed and Taspase1-processed TFIIA. How precisely Taspase1 processing affects TFIIA functions is not understood. Here we report that Taspase1 processing alters TFIIA interactions with TFIID and the conformation of TFIID/TFIIA promoter complexes. We further show that Taspase1 processing induces increased sensitivity of TFIID/TFIIA complexes to the repressor NC2, which is counteracted by the presence of an INR core promoter element. Our results provide first evidence that Taspase1 processing affects TFIIA regulation of TFIID and suggest that Taspase1 processing of TFIIA is required to establish INR-selective core promoter activity in the presence of NC2.

Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation.

Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters--one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor--and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors--Dref and Trl--that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer-core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.

Isolation and activity of the promoters for STAT1 and 2 in Atlantic salmon Salmo salar

Signal Transducer and Activator of Transcription (STAT) 1 and 2 molecules are part of the interferon (IFN) type I and type II (γIFN) signalling pathways, key pathways in the innate immune response. Genomic sequence regions upstream from the 5-prime Salmo salar ORFs were obtained and shown to have functional activity through their incorporation into luciferase reporter constructs and subsequent activation by salmonid alpha virus (SAV). The STAT1 and STAT2 putative promoter regions were also induced by co-transfected plasmids expressing γIFN and IFN type I respectively.Two IFN-induced gene regulatory motifs (GAAANN) associated in a complete Interferon Stimulating Response Element (ISRE) were identified in the STAT1 putative promoter sequence and several GAS elements conforming to Boehm's consensus TTNCNNNAA. Sixteen IFN-induced gene regulatory motifs (GAAANN) could be identified in the STAT2 putative promoter region but no Boehm's GAS element nor ISRE. A palindromic sequence that conforms to Decker's consensus GAS element TTCNNN(N)GAA was identified. The reporter constructs generated here may prove an additional tool for refining knowledge on interferon signalling in fish and the inhibition of such by some fish viral pathogens.

Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP.

In vitro transcription profiling of the σ S subunit of bacterial RNA polymerase: re-definition of the σ S regulon and identification of σ S -specific promoter sequence elements

Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ70 (the housekeeping σ subunit) and σS (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ70, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the −35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the −10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the −10/+1 region could improve promoter recognition by EσS.

Dynamic reprogramming of chromatin accessibility during Drosophilaembryo development

BackgroundThe development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology.ResultsWe used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm.ConclusionsOur results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features.

The use of 35S and Tnos expression elements in the measurement of genetically engineered plant materials

An online survey was conducted by the International Life Sciences Institute, Food Biotechnology Committee, on the use of qualitative and quantitative polymerase chain reaction (PCR) assays for cauliflower mosaic virus 35S promoter and Agrobacterium tumefaciens Tnos DNA sequence elements for the detection of genetically engineered (GE) crop plant material. Forty-four testing laboratories around the world completed the survey. The results showed the widespread use of such methods, the multiplicity of published and in-house methods, and the variety of reference materials and calibrants in use. There was an interest on the part of respondents in validated quantitative assays relevant to all GE events that contain these two genetic elements. Data are presented by testing two variations each of five published real-time quantitative PCR methods for 35S detection on eight maize reference materials. The results showed that two of the five methods were not suitable for all the eight reference materials, with poor linear regression parameters and multiple PCR amplification products for some of the reference materials. This study demonstrates that not all 35S methods produce satisfactory results, emphasizing the need for method validation.

U3 snoRNA promoter reflects the RNA’s function in ribosome biogenesis

An efficiently expressed "tagged" gene system was used to study promoter sequence elements in genes encoding the U3 snoRNAs of Schizosaccharomyces pombe. Transcription was found dependent on two previously thought, mutually exclusive elements, a TATA-box, as found in other S. pombe snRNA gene promoters, and a Homol D-box, often considered a TATA-box analogue in the promoters of genes that encode ribosomal proteins. The Homol D-box is critical while the TATA-box strongly influences transcription efficiency but is not essential. The results suggest that the U3 snoRNA promoter may represent the fusion of two promoter systems reflecting the special role of this RNA in ribosome biogenesis. Steady state measurements of cellular RNAs support such a coordinated regulation of the U3 snoRNA.