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General transcription factors and subunits of RNA polymerase III
Abstract
In the course of evolution of multi-cellular eukaryotes, paralogs of general transcription factors and RNA polymerase subunits emerged. Paralogs of transcription factors and of the RPC32 subunit of RNA polymerase III play important roles in cell type- and promoter-specific transcription. Here we discuss their respective functions.
... Pol III initiates transcription from 3 types of promoters. Each promoter type is used to recruit the polymerase to a specific class of genes and requires a particular combination of TFs [9,41] (Fig 4A) required for the expression of 5S (type 1 promoter), TFIIIC for expression of 5S and tRNAs (types 1 and 2), while SNAPc is required for expression of other RNAs such as 7SK [9,41] (type 3). To directly examine which Pol III targets contribute to ageing, we decide to manipulate TFs that are required for initiation by the polymerase, as this allowed us to restrict the classes of genes whose expression is affected. ...
... Pol III initiates transcription from 3 types of promoters. Each promoter type is used to recruit the polymerase to a specific class of genes and requires a particular combination of TFs [9,41] (Fig 4A) required for the expression of 5S (type 1 promoter), TFIIIC for expression of 5S and tRNAs (types 1 and 2), while SNAPc is required for expression of other RNAs such as 7SK [9,41] (type 3). To directly examine which Pol III targets contribute to ageing, we decide to manipulate TFs that are required for initiation by the polymerase, as this allowed us to restrict the classes of genes whose expression is affected. ...
tRNAs are evolutionarily ancient molecular decoders essential for protein translation. In eukaryotes, tRNAs and other short, noncoding RNAs are transcribed by RNA polymerase (Pol) III, an enzyme that promotes ageing in yeast, worms, and flies. Here, we show that a partial reduction in Pol III activity specifically disrupts tRNA levels. This effect is conserved across worms, flies, and mice, where computational models indicate that it impacts mRNA decoding. In all 3 species, reduced Pol III activity increases proteostatic resilience. In worms, it activates the unfolded protein response (UPR) and direct disruption of tRNA metabolism is sufficient to recapitulate this. In flies, decreasing Pol III’s transcriptional initiation on tRNA genes by a loss-of-function in the TFIIIC transcription factor robustly extends lifespan, improves proteostatic resilience and recapitulates the broad-spectrum benefits to late-life health seen following partial Pol III inhibition. We provide evidence that a partial reduction in Pol III activity impacts translation, quantitatively or qualitatively, in both worms and flies, indicating a potential mode of action. Our work demonstrates a conserved and previously unappreciated role of tRNAs in animal ageing.
... Chez l'homme, on trouve deux isoformes de l'ARN Pol III selon qu'elle contienne la sous-unité RPC32α (ARN Pol IIIα) ou RPC32β (ARN Pol IIIβ). La forme β est considérée comme la forme générale de l'ARN Pol III, la forme α étant exprimée dans des types cellulaires spécifiques et de manière intéressante dans les cellules indifférenciées (Haurie et al., 2010 ;Teichmann et al., 2010). ...
... On notera l'existence chez les mammifères de trois paralogues de TBP : les TBP-related factors (TRF). Parmi eux, TRF1 et TRF3 sont vraisemblablement les seules formes impliquées dans la transcription par l'ARN Pol III (pour revue, Teichmann et al., 2010). ...
RNA polymerase III synthetizes many small untranslated RNA, including tRNA and 5S rRNA which are essential to cell growth. In this work, we took an interest in RNA polymerase III transcription regulation in the baker’s yeast, Saccharomyces cerevisiae. We have detected Sub1 on all class III genes in vivo. We also observed that Sub1 is able to stimulate RNA polymerase III transcription which has been reconstituted in vitro with TFIIIB et TFIIIC recombinants factors and purified RNA polymerase III. Sub1 stimulates two steps of RNA polymerase III transcription : initiation and facilitated reinitiation. Supplementary experiments established that Sub1 directly interacts with TFIIIB and TFIIIC transcription factors. Finally, we showed that Sub1 deletion in yeast leads to a decrease in RNA polymerase III transcription during exponential phase. Then, we tried to determine which link could exist between Sub1, the activator, and Maf1, the repressor of RNA polymerase III transcription. Furthermore, we attempted to identify other elements which could interact with Sub1 during transcription regulation.
... Based on the above summarized information, the cis-and transacting elements taking part in Pol III-dependent transcription appear to constitute a simple and well-defined tool kit, uniformly exploited in all eukaryotic cells for the production of predominantly housekeeping RNAs. In spite of this apparent simplicity, however, studies in the last decade have significantly expanded the inventory of known class III genes, the set of trans-acting components known to participate in Pol III transcription, and the number of known Pol III regulatory mechanisms during cell growth and differentiation, as detailed in recent reviews Dumay-Odelot et al., 2010;Teichmann et al., 2010). Concomitantly, our knowledge of Pol III cis-regulatory elements and of their occurrence and activity in eukaryotic genomes has also increased. ...
... These findings anticipated the crucial discovery of TBP as a universal core TF, participating in the inner workings of all the three eukaryotic transcription machineries (Hernandez, 1993;Rigby, 1993;White et al., 1992), a property that was also already known for five subunits shared by the three nuclear RNA polymerases (Carles et al., 1991). Since then, the sharing of components by the three transcription systems, or the presence of paralogous polypeptides playing similar functions in different systems, has perhaps become less surprising, but not less challenging in terms of mechanistic interpretation (Carter and Drouin, 2010;Geiger et al., 2010;Kassavetis et al., 2010;Lefevre et al., 2011;Teichmann et al., 2010). As significant examples, we mention in particular the Pol II elongation factor TFIIS and the Pol II coactivator protein Sub1, that have recently been located at Pol IIItranscribed genes in S. cerevisiae by genome-wide studies, but whose function in transcription of these genes is only partially understood (Ghavi-Helm et al., 2008;Rosonina et al., 2009;Tavenet et al., 2009). ...
Eukaryotic genomes are punctuated by a multitude of tiny genetic elements, that share the property of being recognized and transcribed by the RNA polymerase (Pol) III machinery to produce a variety of small, abundant non-protein-coding (nc) RNAs (tRNAs, 5S rRNA, U6 snRNA and many others). The highly selective, efficient and localized action of Pol III at its minute genomic targets is made possible by a handful of cis-acting regulatory elements, located within the transcribed region (where they are bound by the multisubunit assembly factor TFIIIC) and/or upstream of the transcription start site. Most of them participate directly or indirectly in the ultimate recruitment of TFIIIB, a key multiprotein initiation factor able to direct, once assembled, multiple transcription cycles by Pol III. But the peculiar efficiency and selectivity of Pol III transcription also depends on its ability to recognize very simple and precisely positioned termination signals. Studies in the last few years have significantly expanded the set of known Pol III-associated loci in genomes and, concomitantly, have revealed unexpected features of Pol III cis-regulatory elements in terms of variety, function, genomic location and potential contribution to transcriptome complexity. Here we review, in a historical perspective, well established and newly acquired knowledge about Pol III transcription control elements, with the aim of providing a useful reference for future studies of the Pol III system, which we anticipate will be numerous and intriguing for years to come.
... Corresponding to the map position of 6ofCl we identified a major ORF which would encode a polypeptide of 170 amino acid residues (19295 Da). This ORF was preceded by a putative ribosome-binding site and sequences that corresponded to the -35 (CGgtTAA) and -10 (CAAACTAA) consensus sequence of EaF-and EaG-recognized promoters (Moran, 1993). Immediately following this ORF was a region of dyad symmetry followed by a stretch of T residues that could function as a rho-independent transcriptional terminator. ...
... Following 6ofC is the 5' end of an ORF, designated ORF4, which extends to the HindIII(, site. This ORF is preceded by a putative ribosome-binding site and sequences (aaGACt and TAcAAT) that could correspond to the -35 and -10 consensus sequence of promoters recognized by the vegetative RNA polymerase, EaA (Moran, 1993). The 105 codons of ORF4 we have identified would encode a polypeptide that has significant homology to the 5' region of the ruuA gene of E. cofi. ...
A mutation, bofC1, that restores sigma K activation in Bacillus subtilis strains unable to produce active sigma G has been identified. This mutation defines a new sporulation gene, bofC, that has been cloned and sequenced and encodes a 19 kDa protein. bofC is transcribed in the forespore by RNA polymerase associated with the transcription factors sigma F (E sigma F) and sigma G (E sigma G). BofC acts negatively on SpoIVB and the results described suggest that BofC regulates SpoIVB activity and its intercompartmental signalling role in the sigma K checkpoint.
... Type III are distinct from Type I and II promoters as they do not require TFIIIC for Pol III mediated transcription but employ SNAPc, a TF also associated with Pol II transcription (Schramm and Hernandez, 2002). This general set of promoter types and TFs is essentially conserved across wide evolutionary distances but with some phyla-specific differences (Schramm and Hernandez, 2002;Teichmann et al., 2010). Additional TFs regulating Pol III activity include Myc, a transcriptional activator that can act on all three Pols (Gomez-Roman et al., 2003;Campbell and White, 2014), as well as the protein Maf1, a highly conserved repressor of Pol III activity (Upadhya et al., 2002;Vorländer et al., 2020). ...
Transcription in eukaryotic cells is performed by three RNA polymerases. RNA polymerase I synthesises most rRNAs, whilst RNA polymerase II transcribes all mRNAs and many non-coding RNAs. The largest of the three polymerases is RNA polymerase III (Pol III) which transcribes a variety of short non-coding RNAs including tRNAs and the 5S rRNA, in addition to other small RNAs such as snRNAs, snoRNAs, SINEs, 7SL RNA, Y RNA, and U6 spilceosomal RNA. Pol III-mediated transcription is highly dynamic and regulated in response to changes in cell growth, cell proliferation and stress. Pol III-generated transcripts are involved in a wide variety of cellular processes, including translation, genome and transcriptome regulation and RNA processing, with Pol III dys-regulation implicated in diseases including leukodystrophy, Alzheimer’s, Fragile X-syndrome and various cancers. More recently, Pol III was identified as an evolutionarily conserved determinant of organismal lifespan acting downstream of mTORC1. Pol III inhibition extends lifespan in yeast, worms and flies, and in worms and flies acts from the intestine and intestinal stem cells respectively to achieve this. Intriguingly, Pol III activation achieved through impairment of its master repressor, Maf1, has also been shown to promote longevity in model organisms, including mice. In this review we introduce the Pol III transcription apparatus and review the current understanding of RNA Pol III’s role in ageing and lifespan in different model organisms. We then discuss the potential of Pol III as a therapeutic target to improve age-related health in humans.
... To verify these results, a second approach was used to repress RNA pol III-dependent transcription. Downregulation of BRF1, an RNA pol III-specific TFIIIB transcription factor subunit (45), also produced a decrease in cell viability and an increase in cleaved caspase-3 expression in the presence of doxorubicin ( Fig. 6C-D). We also confirmed our results using MAF1-deficient (Maf1 −/− ) mouse embryonic fibroblast cells (MEFs). ...
MAF1 homolog, negative regulator of RNA polymerase III (MAF1) is a key repressor of RNA polymerase (pol) III-dependent transcription and functions as a tumor suppressor. Its expression is frequently down-regulated in primary human hepatocellular carcinomas (HCCs). However, this reduction in MAF1 protein levels does not correlate with its transcript levels, indicating that MAF1 is regulated posttranscriptionally. Here, we demonstrate that MAF1 is a labile protein whose levels are regulated through the ubiquitin-dependent proteasome pathway. We found that MAF1 ubiquitination is enhanced upon mTOR complex 1 (TORC1)-mediated phosphorylation at Ser-75. Moreover, we observed that the E3 ubiquitin ligase cullin-2 (CUL2) critically regulates MAF1 ubiquitination and controls its stability and subsequent RNA pol III-dependent transcription. Analysis of the phenotypic consequences of modulating either CUL2 or MAF1 protein expression revealed changes in actin cytoskeleton reorganization and altered sensitivity to doxorubicin-induced apoptosis. Repression of RNA pol III-dependent transcription by chemical inhibition or knockdown of BRF1 RNA pol III transcription initiation factor subunit (BRF1) enhanced HCC cell sensitivity to doxorubicin, suggesting that MAF1 regulates doxorubicin resistance in HCC by controlling RNA pol III-dependent transcription. Together, our results identify the ubiquitin proteasome pathway and CUL2 as important regulators of MAF1 levels. They suggest that decreases in MAF1 protein underlie chemoresistance in HCC and perhaps other cancers and point to an important role for MAF1 and RNA pol III-mediated transcription in chemosensitivity and apoptosis.
... The high level of crystal protein synthesis in Bt and its co-ordination with the stationary phase are controlled by a variety of mechanisms occurring at the transcriptional, post transcriptional and post translational levels. In Bt, most of the cry genes are expressed only during sporulation; few genes are expressed during the vegetative phase (Moran 1993 ). ...
Bacillus thuringiensis (Bt) is used to control agriculturally-important pests. It is a Gram positive spore-forming bacterium which produces parasporal proteinaceous inclusions during the sporulation phase. These crystalline parasporal inclusions are toxic to a wide spectrum of insects including the orders Lepidoptera, Coleopteran, Diptera, etc. The Bt insecticide proteins are toxic only after ingestion by the susceptible insects. The main steps involved when the Cry protein is ingested by the insect is comprised of solubilization of the protoxin, its enzymatic activation by terminal cleavage, receptor binding in brush border membrane of the midgut, pore formation, consequent disruption of ionic potential and destruction of the epithelial membrane leading to cell death. The first discovery of Bt was in 1901 when Ishiwata discovered a bacterium in Japan and in 1915, Berliner in Germany renamed it as Bacillus thuringiensis. Following a brief introduction, this chapter addresses the classification, the general structure of Cry toxin, its mode of action, strategies to improve the insecticidal activity of Cry proteins, transgenic plants developed using Bt genes, resistance to Bt toxins and resistance management, and an overall brief account of Bt and its insecticidal proteins, from 1901 to the present.
... Earlier studies have shown that Tfc7 is required for RNA polymerase (Pol) III in transcribing 5S rRNA, which is a part of ribosomes (Teichmann et al., 2010;Acker et al., 2013). In S. cerevisiae, Tfc7 is essential. ...
... Notably, PIC formation and transcriptional activation is not uniform in eukaryotes either and the concerted action of TBP and TFB in analogy to S. acidocaldarius is characteristic for the RNAPIII transcription system as well. RNAPIII-directed transcription is aided by two multiprotein factors termed TF(III)B and TF(III)C (67). TF(III)B is composed of TBP, the TF(II)B-factor related factor and B double prime (Figure 7). ...
During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein
(TBP). Subsequent association of transcription factor B (TFB) with the TBP–DNA complex is followed by the recruitment of the
ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved
in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions.
Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in
the absence and presence of TFB in real-time. We observed that the lifetime of the TBP–DNA interaction differs significantly
between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear,
stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes
the fully bent TBP–promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP–DNA
interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the
way to the formation of the pre-initiation complex.
... Yeast Pol III consists of 17 subunits which have structural and functional homologs in human cells. Transcription of tRNA genes requires the multisubunit initiation factors TFIIIB and TFIIIC which specifically bind to internal control regions [1,2]. ...
Yeast Fba1 (fructose 1,6-bisphosphate aldolase) is a glycolytic enzyme essential for viability. The overproduction of Fba1 enables the overcoming of a severe growth defect caused by a missense mutation rpc128-1007 in a gene encoding the C128 protein, the second largest subunit of the RNA polymerase III complex. The suppression of the growth phenotype by Fba1 is accompanied by enhanced de novo tRNA transcription in rpc128-1007 cells. We inactivated residues critical for the catalytic activity of Fba1. Overproduction of inactive aldolase still suppressed the rpc128-1007 phenotype, indicating that function of this glycolytic enzyme in RNA polymerase III transcription is independent of its catalytic activity. Yeast Fba1 was determined to interact with the RNA polymerase III complex by coimmunoprecipitation. Additionally, a role of aldolase in control of tRNA transcription was confirmed by ChIP experiments. The results indicate a novel direct relationship between RNA polymerase III transcription and aldolase.
... It will also be of particular interest to investigate the possible role in facilitated recycling of the Rpc31 subunit (yeast nomenclature; RPC32 in humans), previously shown to be involved in transcription initiation [106]. Intriguingly, it appears to be the only subunit having no paralog in Pol I and Pol II transcription machineries, and it has been shown to exist in two differentially expressed isoforms in human cells, suggesting highly specialized functions for this subunit [107,108]. ...
The retention of transcription proteins at an actively transcribed gene contributes to maintenance of the active transcriptional state and increases the rate of subsequent transcription cycles relative to the initial cycle. This process, called transcription reinitiation, generates the abundant RNAs in living cells. The persistence of stable preinitiation intermediates on activated genes representing at least a subset of basal transcription components has long been recognized as a shared feature of RNA polymerase (Pol) I, II and III-dependent transcription in eukaryotes. Studies of the Pol III transcription machinery and its target genes in eukaryotic genomes over the last fifteen years, has uncovered multiple details on transcription reinitiation. In addition to the basal transcription factors that recruit the polymerase, Pol III itself can be retained on the same gene through multiple transcription cycles by a facilitated recycling pathway. The molecular bases for facilitated recycling are progressively being revealed with advances in structural and functional studies. At the same time, progress in our understanding of Pol III transcriptional regulation in response to different environmental cues points to the specific mechanism of Pol III reinitiation as a key target of signaling pathway regulation of cell growth. This article is part of a Special Issue entitled: Transcription by Odd Pols.
... The sequences of TFIIIC-bound A and B boxes and of the SNAPc-bound PSE, are rather degenerated in sequence (especially the A box and the PSE). However, the position of the A box downstream of the TSS (~8 bp) is strongly conserved, as are the positions of the PSE and of the TATA element upstream of the TSS (60-65 and 30 bp, respectively) [56,86]. The nature, types and roles of Pol III transcription control elements have been the subject of a recent review, to which the readers are invited to refer for more complete information [4] (see also Fig. 1). ...
The RNA polymerase (Pol) III transcription system is devoted to the production of short, generally abundant noncoding (nc) RNAs in all eukaryotic cells. Previously thought to be restricted to a few housekeeping genes easily detectable in genome sequences, the set of known Pol III-transcribed genes (class III genes) has been expanding in the last ten years, and the issue of their detection, annotation and actual expression has been stimulated and revived by the results of recent high-resolution genome-wide location analyses of the mammalian Pol III machinery, together with those of Pol III-centered computational studies and of ncRNA-focused transcriptomic approaches. In this article, we provide an outline of distinctive features of Pol III-transcribed genes that have allowed and currently allow for their detection in genome sequences, we critically review the currently practiced strategies for the identification of novel class III genes and transcripts, and we discuss emerging themes in Pol III transcription regulation which might orient future transcriptomic studies. This article is part of a Special Issue entitled: Transcription by Odd Pols.
... In contrast, type III promoters are only present in vertebrates (e.g. H. sapiens U6 snRNA) and utilize invariably a canonical TATA box as well as a further upstream sequence called proximal sequence element (PSE), which is recognized by the multi-subunit transcription factor SNAPc. SNAPc and TFIIIB bind cooperatively to the PSE and the TATA box, respectively, culminating in efficient Pol III recruitment [39,40,57]. ...
RNA polymerase I and III are responsible for the bulk of nuclear transcription in actively growing cells and their activity impacts the cellular biosynthetic capacity. As a consequence, RNA polymerase I and III deregulation has been directly linked to cancer development. The complexity of RNA polymerase I and III transcription apparatuses has hampered their structural characterization. However, in the last decade tremendous progresses have been made, providing insights into the molecular and functional architecture of these multi-subunit transcriptional machineries. Here we summarize the available structural data on RNA polymerase I and III, including specific transcription factors and global regulators. Despite the overall scarcity of detailed structural data, the recent advances in the structural biology of RNA polymerase I and III represent the first step towards a comprehensive understanding of the molecular mechanism underlying RNA polymerase I and III transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols.
... Two successive stem loop structures (positions 3047 to 3080 and 3081 to 3117, DG7181 kJ and 7119 kJ, respectively) that are potential rho-independent transcription terminators are present downstream of xynD and of ORF3 (Fig. 1). Sequences that resemble s A consensus sequences of B. subtilis [26] are present upstream of xynD and ORF5 (xylA), and between the divergently transcribed ORFs 3 and 4. Classic studies by Dehority and coworkers [1,6] showed that not all cellulolytic R. flavefaciens strains can utilise xylan breakdown products. R. flavefaciens strains B1a, B34b, C1a and C94 all utilised cellulose from intact forages such as bromegrass and alfalfa, but hemicellulose utilisation, measured by loss of total pentose sugar, varied dramatically with strains C1a and B34b able to utilise little or no detectable pentose, whereas B1a and C94 utilised significant pentose [6]. ...
Strains of the rumen cellulolytic bacterium Ruminococcus flavefaciens vary in their ability to utilise isolated plant xylans for growth. Here an 11.5 kb fragment of genomic DNA from the xylan-utilizing R. flavefaciens strain 17 that contains the xynD gene, which encodes an extracellular xylanase/β -(1,3-1,4)-glucanase, was analysed. Sequencing revealed five consecutive open reading frames downstream from xynD on the same strand, preceded by the divergently transcribed ORF3. These include the following genes likely to be involved in utilisation of xylan breakdown products: xylA, encoding a β -(1,4)-xylosidase, xsi, encoding a xylose isomerase and ORF8 encoding part of an ABC-type sugar transporter. The products of ORF3 and of a partial ORF1 found upstream of xynD, show significant sequence similarity to AraC-type regulatory proteins while ORF4 and ORF7 show no close relationship to other known proteins. Homologues of the xylA and xsi genes, and inducible β -xylosidase activity, were readily detectable in three xylan-utilizing R. flavefaciens strains 17, B1a and C94 but not in two xylan non-utilizing strains, C1a and B34b, suggesting that this cluster may be absent from xylan non-utilizing strains.
... TBP and two RNAP-III-specific TAFs (BrfI and BdpI) assemble into the TFIIIB complex, which is involved in RNAP-III transcription. In vertebrates, there are two versions of TFIIIB complex containing either BrfI or its homolog BrfII (reviewed in [16]). Besides TFIIIB, TBP also interacts with the snRNA activating protein complex (SNAPc) at the human U6 promoter [17]. ...
ABSTRACT: TATA binding protein (TBP) is a key component of the eukaryotic transcription initiation machinery. It functions in several complexes involved in core promoter recognition and assembly of the pre-initiation complex. Through gene duplication eukaryotes have expanded their repertoire of TATA binding proteins, leading to a variable composition of the transcription machinery. In vertebrates this repertoire consists of TBP, TBP-like factor (TLF, also known as TBPL1, TRF2) and TBP2 (also known as TBPL2, TRF3). All three factors are essential, with TLF and TBP2 playing important roles in development and differentiation, in particular gametogenesis and early embryonic development, whereas TBP dominates somatic cell transcription. TBP-related factors may compete for promoters when co-expressed, but also show preferential interactions with subsets of promoters. Initiation factor switching occurs on account of differential expression of these proteins in gametes, embryos and somatic cells. Paralogs of TFIIA and TAF subunits account for additional variation in the transcription initiation complex. This variation in core promoter recognition accommodates the expanded regulatory capacity and specificity required for germ cells and embryonic development in higher eukaryotes.
... Analysis of the DNA sequence upstream from the start site revealed the presence of a putative A -like consensus sequence , ATGAATN 17 GATAAA (Fig. 2 ). This sequence represents a total of 7 of 12 possible matches to the Bacillus subtilis A consensus sequence (TTGACAN 17 TATAAT [33] ). In addition , this primer extension analysis indicated that the apparent transcription start site was the same in exponential-and stationary-phase cells (Fig. 5, lanes 1 and 2). ...
Previously in our laboratory, a PCR-based strategy was used to isolate potential sensor gene fragments from the Staphyloccus aureus genome. One DNA fragment was isolated that shared strong sequence similarity to genes encoding bacterial sensor proteins, indicating that it originated from within a potential staphylococcal sensor protein gene. In this study, the DNA surrounding the PCR product origin was cloned and sequenced. This analysis revealed the presence of two genes, termed lytS and lytR, whose deduced amino acid sequences were similar to those of members of the two-component regulatory system family of proteins. S. aureus cells containing an insertional disruption of lytS exhibited a marked propensity to form aggregates in liquid culture, suggesting that alterations in cell surface components exist in this strain. Transmission electron microscopic examination of these cells revealed that the cell surface was rough and diffuse and that a large proportion of the cell population had lysed. The lytS mutant also exhibited increased autolysis and an altered level of murein hydrolase activity produced compared with the parental strain, NCTC 8325-4. These data suggest that the lytS and lytR gene products control the rate of autolysis in S. aureus by affecting the intrinsic murein hydrolase activity associated with the cell.
... Indeed, if there was no synthesis, P-galactosidase specific activity would drop rapidly as in the case of the spollD-IacZ fusion after t,. These results suggest that there are two phases in cr_yllIA expression in Bt cells : an activation period which ends concomitantly with the turning on of oE and a period during which c r~y l l l A expression is maintained (roughly until oK becomes functional in the mother-cell compartment, i.e. stage IV of sporulation) (Ribier & Lecadet, 1973;Bechtel & Bulla, 1976 ;Errington, 1993 ;Moran, 1993). ...
The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK). cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.
... Programmed switches in gene expression can be realized by a number of different mechanisms, including phosphorylation cascades, repressor/activator circuits and changes in the concentrations of small effector molecules, but one of the theoretically simplest mechanisms is the use of alternative sigma factors to alter RNA polymerase (RNAP) core specificity. In Bacillus subtilis, the developmental pathway leading to sporulation involves at least five different sigma factors in addition to the housekeeping sigma factor Hoch, 1993;Moran, 1993). In Escherichia coli, seven different sigma factors have been identified, and at least four of them are directly involved in the cell's response to stress conditions; two sigma factors are involved in the heat shock response, one is involved in nitrogen regulation and the fourth is important in stationary phase (reviewed in Gross et al., 1992). ...
A mutation in the Escherichia coli gene encoding the stationary phase-inducible sigma factor (sigmaS, RpoS) not only abolishes transcription of some genes in stationary phase, but also causes superinduction of other stationary phase-induced genes. We have examined this phenomenon of repression by sigmaS using as a model system the divergently transcribed stationary phase-inducible genes, uspA and uspB. uspA is transcribed by sigma70-programmed RNA polymerase and is superinduced in an rpoS mutant, while uspB induction is sigmaS dependent. The data suggest that the superinduction of uspA is caused by an increased amount of sigma70 bound to RNA polymerase in the absence of the competing sigmaS. Increasing the ability of sigma70 to compete against sigmaS by overproducing sigma70 mimics the effect of an rpoS mutation by causing superinduction of sigma70-dependent stationary phase-inducible genes (uspA and fadD), silencing of sigmaS-dependent genes (uspB, bolAp1 and fadL) and inhibiting the development of sigmaS-dependent phenotypes, such as hydrogen peroxide resistance in stationary phase. In addition, overproduction of sigmaS markedly reduced stationary phase expression of a sigma70-dependent promoter. Thus, we conclude that sigma factors compete for a limiting amount of RNA polymerase during stationary phase. The implications of this competition in the passive control of promoter activity is discussed.
... Its start was found to lie 5 nt downstream from a putative -10 box identified in the sequence (TTAAAT). Upstream from this box separated by 18 nt is a -35 box (ATGATA) with sequence similar to the consensus sequence TTGACA that is typical of B. subtilis sigmaA-dependent promoters [11]. ...
Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis.
Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling.
A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane).
... At t 0 and t 2 , a transcript was detected with its 5Ј end 267 bp upstream from the presumed inhA2 start codon (Fig. 2B). The putative Ϫ10 and Ϫ35 boxes of the inhA2 promoter ( Fig. 2A) resemble the A promoter consensus (TTGACA, 16 to 18 bases, TATAAT) of Bacillus subtilis (39). ...
The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts.
We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry−. The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain
(HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2′-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence
of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single
and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of
the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential
for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity
of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of ΔinhA2 mutant strain were ineffective in providing synergism whereas those of the ΔinhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.
RNA polymerase (Pol) III transcribes small untranslated RNAs such as 5S ribosomal RNA, transfer RNAs, and U6 small nuclear RNA. Because of the functions of these RNAs, Pol III transcription is best known for its essential contribution to RNA maturation and translation. Surprisingly, it was discovered in the last decade that various inherited mutations in genes encoding nine distinct subunits of Pol III cause tissue-specific diseases rather than a general failure of all vital functions. Mutations in the POLR3A, POLR3C, POLR3E and POLR3F subunits are associated with susceptibility to varicella zoster virus-induced encephalitis and pneumonitis. In addition, an ever-increasing number of distinct mutations in the POLR3A, POLR3B, POLR1C and POLR3K subunits cause a spectrum of neurodegenerative diseases, which includes most notably hypomyelinating leukodystrophy. Furthermore, other rare diseases are also associated with mutations in genes encoding subunits of Pol III (POLR3H, POLR3GL) and the BRF1 component of the TFIIIB transcription initiation factor. Although the causal relationship between these mutations and disease development is widely accepted, the exact molecular mechanisms underlying disease pathogenesis remain enigmatic. Here, we review the current knowledge on the functional impact of specific mutations, possible Pol III-related disease-causing mechanisms, and animal models that may help to better understand the links between Pol III mutations and disease.
LTR-retrotransposons are widespread transposable elements in eukaryotes. Like retroviruses, they replicate by reverse transcription of their RNA into cDNA, which is integrated into the host genome by their own integrase (IN). High-throughput sequencing studies clearly established that integration does not occur randomly throughout the host-cell genome. Deep insights on retroviral biology have been gained by their study in yeast using the Ty1 LTR-retrotransposon as a working model. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of class III genes, the genes transcribed by RNA polymerase III (Pol III). Recent data revealed the importance of AC40, a Pol III subunit in this targeting. An interaction between the Ty1 IN and AC40 is necessary for integration site choice at the Pol III genes. Nevertheless, the molecular mechanism remains largely unknown. To obtain a global view of the entire phenomenon that occurs on the integration site we would like to exhaustively determine the proteins that interact with Ty1 IN and analyze their role in both Ty1 integration and RNA Pol III transcription. To achieve this goal, we have developed proteomic approaches to identify new Ty1 integrase cellular partners. We have identified several novel Ty1 IN partners that seem interesting and their molecular role in Ty1 retrotransposition will be studied. However, in the tenure of my PhD, I have particularly worked to decipher the molecular role of the casein kinase II protein in Ty1 retrotransposition.
In eukaryotes, RNA polymerase (RNAP) III transcribes the tRNAs, the 5S ribosomal RNA and a half a dozen known untranslated RNA. Mammalian genome contains several thousand of repeated elements, the Short interspersed repetitive elements (SINE). In vitro, they are transcribed by RNAP III. RNAP III transcription levels determine cell growth and proliferation and, importantly, its deregulation is associated with cancer. Looking at the genome-wide distribution of RNAP III and its transcription factors, TFIIIB and TFIIIC, we develop a highly specific tandem ChIP-sequencing method. We have determined the set of genes that are transcribed by RNAP III in mouse embryonic stem cells. We discovered that not all known class III genes were transcribed in ES cells. We also observed that RNAP III and its transcription factors were present at thirty unannotated sites on the mouse genome, only one of which was conserved in human. Only a couple of hundreds of SINEs out of more than half a million are associated with RNAP III in mouse ES cells. Our study reveals numerous 'TFIIIC-only' sites, called ETC for extra-TFIIIC loci in yeast. These sites are correlated with association of CTCF and the cohesin. Cohesin has been shown to occupy sites bound by CTCF and to contribute to DNA loop formation associated with gene repression or activation. This observation suggests that TFIIIC may play a role in chromosome organization in mouse. We also demonstrated that TCEA1, the ubiquitous isoform of TFIIS RNAP II elongation factor, is associated with active class III genes suggesting that TFIIS is a RNAP III transcription factor in mammals. Finally, the distribution of TFIIS on RNAP II-transcribed genes indicated that its recruitment does not control the transition of RNAP II paused at genes 5' end into elongation.
SIRT1, member of the sirtuins family, is an NAD-dependent deacetylase, playing an essential role in controlling gene expression. In addition to modifying histones, SIRT1 can affect the activity of several transcription factors and their target genes. A fundamental question is to understand the molecular mechanisms by which SIRT1 controls the expression of genesinvolved in cell proliferation and energy metabolism. To identify protein partners of SIRT1, we used the method of TAP-TAG purification from a soluble nuclear fraction and a chromatin anchored fraction of Mef cells stably expressing ectopic copy of SIRT1 (SIRT1-e). We were able to identify a SIRT1 complex associated with both cell proliferation factor Ki67, and TFIIIC,subunit required for assembly of the RNA polymerase III pre-initiation complex. By deleting Sirt1, and by specifically inhibiting Ki67 expression, we showed that the RNA Polymerase III transcription machinery and cell proliferation were strongly affected. All of my results clearly shows that SIRT1, Ki67, and TFIIIC are within a same protein complex, SIRT1 and Ki67, acting in coordination to regulate the expression level of SINES and LINES, transcribed from RNA polymerase III transcription machinery.
tRNA synthesis by yeast RNA polymerase III (Pol III) is down-regulated under growth-limiting conditions. This control is mediated by Maf1, a global negative regulator of Pol III transcription. Conserved from yeast to man, Maf1 was originally discovered in Saccharomyces cerevisiae by a genetic approach. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from the recently reported structural and biochemical data on Pol III and Maf1. The phosphorylation status of Maf1 determines its nuclear localization and interaction with the Pol III complex and several Maf1 kinases have been identified to be involved in Pol III control. Moreover, Maf1 indirectly affects tRNA maturation and decay. Here I discuss the current understanding of the mechanisms that oversee the Maf1-mediated regulation of Pol III activity and the role of Maf1 in the control of tRNA biosynthesis in yeast. This article is part of a Special Issue entitled: Transcription by Odd Pols.
RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.
Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5´ and 3´ flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5´ flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a β-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes.
We showed previously that HeLa cell nuclear extracts contain two forms of transcription factor IIIC (TFIIIC) that formed chromatographically distinct TFIIIC-promoter complexes (Hoeffler, W. K., Kovelman, R., and Roeder, R. G. (1988) Cell 53, 907-920). One of these forms, the upper-band form, correlated with TFIIIC transcriptional activity, whereas the lower-band form bound to the VA1 promoter but supported little or no transcriptional activity. Using both transcription and DNA-binding assays, we have now purified both the upper-band form and the lower-band form of TFIIIC to near-homogeneity. The upper-band form is composed of five polypeptides with estimated sizes of 220, 110, 102, 90, and 63 kDa. The largest of these polypeptides can be cross-linked to the VA1 promoter. The lower-band form has a polypeptide structure similar to that of the upper-band form except for the absence or modification of the 110-kDa subunit. Direct assays show that the lower-band form is indeed transcriptionally inactive at all stages of purification, even when assayed with an unfractionated, heat-treated nuclear extract as a complementation system. This inactivity does not result from altered DNA-binding properties; instead, we suggest that the alteration of one of the subunits of TFIIIC renders it unable to interact productively with a downstream component of the transcription complex.
A sigma B-dependent stress gene of Bacillus subtilis was localized downstream of the licS gene. The predicted amino acid sequence exhibited a significant similarity to the sequence of the katE-encoded catalase HPII of Escherichia coli, and we designated it the open reading frame katE. In a B. subtilis katE mutant, catalase 2 could not be detected. The amount of katE-specific mRNA was increased after heat, salt, or ethanol stress or after glucose starvation in a sigma B-dependent manner. As in E. coli, the transcription of the katE gene in B. subtilis was unaffected by the addition of H2O2 to exponentially growing cells. In contrast, the katA gene encoding catalase 1 of B. subtilis showed an induction pattern different from that of katE; katA expression was strongly increased by oxidative stress. The similarity between E. coli sigma S-dependent genes and B. subtilis sigma B-dependent genes suggests that both may confer multiple stress resistance to stationary-phase cells.
A mutation in Bacillus subtilis spo0A codon 97 suppressed the sporulation defect caused by the spo0A9V mutation. The suppressor activity of the codon 97 mutation was evident only in the presence of a novel spo0H allele. Our results suggest that the spo0A gene product interacts with the sigma factor subunit of RNA polymerase.
The 5S gene-specific transcription factor, TFIIIA, was purified approximately 35,000-fold from HeLa cell extracts using a combination of conventional and affinity chromatographic methods. A single polypeptide of apparent molecular mass 42-kDa cofractionates with both 5S DNA binding and 5S transcription activities, and was conclusively identified as human TFIIIA by its ability to direct specific 5S gene transcription in vitro following elution and renaturation from SDS-polyacrylamide gels. The DNase I protection pattern of the purified human factor on a human 5S gene is similar to the pattern previously observed with Xenopus TFIIIA. A Xenopus 5S gene, but not a yeast 5S gene, is able to effectively compete for binding of the human factor. In addition, we report a previously undetected immunological cross-reactivity between human and Xenopus TFIIIA. hTFIIIA is recognized specifically both by polyclonal antisera raised against Xenopus laevis TFIIIA as well as by a monoclonal antibody generated against the amphibian protein. These observations indicate that human TFIIIA is structurally related to the Xenopus oocyte factor and that the previous inability to detect human TFIIIA by immunological methods is due primarily to the low abundance of this factor in HeLa cell extracts.
Four tandem subtilisin-like protease genes were found on a 6,854-bp DNA fragment cloned from the alkalophilic Bacillus sp. strain LG12. The two downstream genes (sprC and sprD) appear to be transcribed independently, while the two upstream genes (sprA and sprB) seem to be part of the same transcript.
A transcriptional analysis of the phosphatidylinositol-specific phospholipase C (plcA) gene of Bacillus thuringiensis indicated that its transcription was activated at the onset of the stationary phase in B. thuringiensis but was not activated in B. subtilis. The B. thuringiensis gene encoding a transcriptional activator required for plcA expression was cloned by using a B. subtilis strain carrying a chromosomal plcA'-'lacZ fusion as a heterologous host for selection. This trans activator (designated PlcR) is a protein of a calculated molecular weight of 33,762 which appears to be distantly related to PreL and NprA, regulator proteins enhancing transcription of neutral protease genes during the stationary phase of a Lactobacillus sp. and B. stearothermophilus, respectively. plcR gene transcription was analyzed in B. thuringiensis and in B. subtilis. PlcR positively regulated its own transcription at the onset of the stationary phase. There is a highly conserved DNA sequence (17 bp) 34 nucleotides upstream from the plcR transcriptional start site and 49 nucleotides upstream from the plcA transcriptional start site. As PlcR positively regulates its own transcription and plcA transcription, this conserved DNA sequence may be the specific recognition target for PlcR activation.
The tre locus from Bacillus subtilis containing the genes treP, treA, and treR has been analyzed for its regulation. We demonstrate that at least treP and treA form an operon whose expression is regulated at the transcriptional level. TreR activity has been investigated in in vivo and in vitro studies. An insertional inactivation of treR led to a constitutive expression of treP and treA. Upstream of treP we identified a 248-bp DNA fragment containing a potential sigmaA-dependent promoter and two palindromes reflecting potential tre operators which led to complex formation with TreR-containing protein extracts in DNA retardation experiments. This complex formation is abolished in the presence of trehalose-6-phosphate, which probably acts as an inducer. Therefore, we assume that treR encodes the specific Tre repressor involved in regulation of the expression of the tre operon.
A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.
The Bacillus subtilis araR locus (mapped at about 294 degrees on the genetic map) comprises two open reading frames with divergently arranged promoters, the regulatory gene, araR, encoding a repressor, and a partially cloned gene, termed araE by analogy to the Escherichia coli L-arabinose permease gene. Here, we report the cloning and sequencing of the entire araE gene encoding a 50.4-kDa polypeptide. The araE gene is monocistronic (as determined by Northern blot analysis), and its putative product is very similar to a number of prokaryotic proton-linked monosaccharide transporters (the group I family of membrane transport proteins). Insertional inactivation of the araE gene leads to a conditional Ara- phenotype dependent on the concentration of L-arabinose in the medium. Therefore, we assume that araE encodes a permease involved in L-arabinose transport into the cell. The araE promoter region contains -10 and -35 regions (as determined by primer extension analysis) very similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigmaA, and the -35 region of the transcription start point for araE is located 2 bp from the -35 region of the araR gene. Transcriptional studies demonstrated that the expression from the araE promoter is induced by L-arabinose, repressed by glucose, and negatively regulated by AraR. These observations are consistent with a model according to which in the absence of L-arabinose, AraR binds to a site(s) within the araE/araR promoter, preventing transcription from the araE promoter and simultaneously limiting the frequency of initiation from its own promoter; the addition of L-arabinose will allow transcription from the araE promoter and increase the frequency of initiation from the araR promoter.
During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.
Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.
Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.
RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.
Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32beta) that led to the demonstration of two human Pol III isoforms (Pol IIIalpha and Pol IIIbeta). RPC32beta-containing Pol IIIbeta is ubiquitously expressed and essential for growth of human cells. RPC32alpha-containing Pol IIIalpha is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32alpha expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32alpha in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation.
The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol
III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally
plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon
domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional
start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially
assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking
analysis places a segment of C53 close to the RNA 3′ end and transcribed DNA strand at the catalytic center of the pol III
elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol
III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II
initiation factor TFIIF.
Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have
implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version
of the popular profile hidden Markov model package. This software is ∼100 times faster than HMMER2 and is more sensitive due
to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described
in detail. Pfam release 24.0 contains 11 912 families, of which a large number have been significantly updated during the
past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/).
Background
TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos.
Results
We find that TBP2 is proteolytically degraded upon meiotic maturation; after germinal vesicle breakdown relatively low levels of TBP2 expression persist. Furthermore, TBP2 localizes to the transcriptionally active loops of lampbrush chromosomes and is recruited to a number of injected promoters in oocyte nuclei. Using an altered binding specificity mutant reporter system we show that TBP2 promotes RNA polymerase II transcription in vivo. Intriguingly, TBP, which in oocytes is undetectable at the protein level, can functionally replace TBP2 when ectopically expressed in oocytes, showing that switching of initiation factors can be driven by changes in their expression. Proteolytic degradation of TBP2 is not required for repression of transcription during meiotic maturation, suggesting a redundant role in this repression or a role in initiation factor switching between oocytes and embryos.
Conclusion
The expression and transcriptional activity of TBP2 in oocytes show that TBP2 is the predominant initiation factor in oocytes, which is substituted by TBP on a subset of promoters in embryos as a result of proteolytic degradation of TBP2 during meiotic maturation.
Development of the germline requires consecutive differentiation events. Regulation of these has been associated with germ cell-specific and pluripotency-associated transcription factors, but the role of general transcription factors (GTFs) remains elusive. TATA-binding protein (TBP) is a GTF involved in transcription by all RNA polymerases. During ovarian folliculogenesis in mice the vertebrate-specific member of the TBP family, TBP2/TRF3, is expressed exclusively in oocytes. To determine TBP2 function in vivo, we generated TBP2-deficient mice. We found that Tbp2(-/-) mice are viable with no apparent phenotype. However, females lacking TBP2 are sterile due to defective folliculogenesis, altered chromatin organization, and transcriptional misregulation of key oocyte-specific genes. TBP2 binds to promoters of misregulated genes, suggesting that TBP2 directly regulates their expression. In contrast, TBP ablation in the female germline results in normal ovulation and fertilization, indicating that in these cells TBP is dispensable. We demonstrate that TBP2 is essential for the differentiation of female germ cells, and show the mutually exclusive functions of these key core promoter-binding factors, TBP and TBP2, in the mouse.
Human 7SK RNA is an abundant 331 nt nuclear transcript generated by RNA polymerase III. Binding of 7SK RNA to HEXIM1/2 turns these proteins into inhibitors of P-TEFb (Positive Transcriptional Elongation Factor b). P-TEFb is required for RNA polymerase II transcription elongation. 7SK RNA is released from P-TEFb/HEXIM/7SK complexes upon an arrest in transcription and physiological stimulations such as cardiac hypertrophy, leading to P-TEFb activation. The released 7SK RNA associates a subset of heterogeneous nuclear ribonucleoproteins (hnRNP). 7SK RNA has been evolutionary conserved in vertebrates and homologues are found in annelid, mollusc and insect genomes. 7SK RNA folds into several hairpins that serve as specific platforms for binding proteins. It is stabilized by mono-methylation of its 5'-triphosphate group and binding of a specific La-Related protein, LARP7 at its 3' end. As the likely best characterized example, 7SK RNA is a paradigm for non-coding RNAs regulating transcription.
Human transcription factor hTFIIIB is necessary to initiate transcription correctly from all RNA polymerase III (pol III) genes which are governed by structurally different promoters, and it is unclear whether hTFIIIB complexes, required for intragenic or 5'-located pol III promoters, are composed of unique or different components. We show here that two different forms of hTFIIIB can be separated physically by ion exchange chromatography. hTFIIIB-alpha shows strong preference for transcription of the U6 over the VAI gene and does not contain TATA binding protein (TBP). After SDS-PAGE and renaturation of proteins, the transcriptional activity of hTFIIIB-alpha can be reconstituted by fractions corresponding to a mean M(r) of 25, 60 and 90 kDa. Upon gradient centrifugation or gel filtration, the activity of hTFIIIB-alpha is associated with an M(r) of 60 +/- 10 kDa, indicating that the components of the complex tend to dissociate. In contrast, hTFIIIB-beta is predominantly active on intragenic pol III promoters. It reveals an M(r) of 300 +/- 30 kDa upon gel filtration and, besides TBP, it contains several associated factors (TAFs). Two of these proteins reveal an M(r) of 60 kDa and 90 kDa, and it is conceivable that they are related to polypeptides of similar mass functionally identified in hTFIIIB-alpha. These protein are probably required for the recruitment of pol III to the initiation site at 5'-located and intragenic promoters.
The TATA-binding protein (TBP)-related factor TRF1, has been described in Drosophila and a related protein, TRF2, has been found in a variety of higher eukaryotes. We report that human (h)TRF2 is encoded by two mRNAs with common protein coding but distinct 5' nontranslated regions. One mRNA is expressed ubiquitously (hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted expression pattern and is extremely abundant in testis. In addition, we show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2. Neither recombinant human (rh)TRF2 nor the native flag.hTRF2-TFIIA complex is able to replace TBP or TFIID in basal or activated transcription from various RNA polymerase II promoters. Instead, rhTRF2, but not the flag.hTRF2-TFIIA complex, moderately inhibits basal or activated transcription in the presence of rhTBP or flag.TFIID. This effect is either completely (TBP-mediated transcription) or partially (TFIID-mediated transcription) counteracted by addition of free TFIIA. Neither rhTRF2 nor flag. hTRF2-TFIIA has any effect on the repression of TFIID-mediated transcription by negative cofactor-2 (NC2) and neither substitutes for TBP in RNA polymerase III-mediated transcription.
Transcription factor IIIB (TFIIIB) is directly involved in transcription initiation by RNA polymerase III in eukaryotes. Yeast contain a single TFIIIB activity that is comprised of the TATA-binding protein (TBP), TFIIB-related factor 1 (BRF1), and TFIIIB", whereas two distinct TFIIIB activities, TFIIIB-alpha and TFIIIB-beta, have been described in human cells. Human TFIIIB-beta is required for transcription of genes with internal promoter elements, and contains TBP, a TFIIIB" homologue (TFIIIB150), and a BRF1 homologue (TFIIIB90), whereas TFIIIB-alpha is required for transcription of genes with promoter elements upstream of the initiation site. Here we describe the identification, cloning, and characterization of TFIIIB50, a novel homologue of TFIIB and TFIIIB90. TFIIIB50 and tightly associated factors, along with TBP and TFIIIB150, reconstitute human TFIIIB-alpha activity. Thus, higher eukaryotes, in contrast to the yeast Saccharomyces cerevisiae, have evolved two distinct TFIIB-related factors that mediate promoter selectivity by RNA polymerase III.
The TATA-binding protein (TBP) is involved in all nuclear transcription. We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III. TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB. A photochemical cross-linking approach was used to survey a collection of human TBP surface residue mutants for their ability to form TFIIIB-DNA complexes reliant on only the TFIIB-related part of Brf1. Mutations impairing complex formation and transcription were identified and mapped on the surface of TBP. The most severe effects were observed for mutations in the C-terminal stirrup of TBP, which is the principal site of interaction between TBP and TFIIB. Structural modeling of the Brf1-TBP complex and comparison with its TFIIB-TBP analog further rationalizes the close resemblance of the TBP interaction with the N-proximal part of Brf1 and TFIIB, and establishes the conserved usage of a TBP surface in pol II and pol III transcription for a conserved function in the initiation of transcription.
TFIIIC in yeast and humans is required for transcription of tRNA and 5 S RNA genes by RNA polymerase III. In the yeast Saccharomyces cerevisiae, TFIIIC is composed of six subunits, five of which are conserved in humans. We report the identification, molecular cloning,
and characterization of the sixth subunit of human TFIIIC, TFIIIC35, which is related to the smallest subunit of yeast TFIIIC.
Human TFIIIC35 does not contain the phosphoglycerate mutase domain of its yeast counterpart, and these two proteins display
only limited homology within a 34-amino acid domain. Homologs of the sixth TFIIIC subunit are also identified in other eukaryotes,
and their phylogenic evolution is analyzed. Affinity-purified human TFIIIC from an epitope-tagged TFIIIC35 cell line is active
in binding to and in transcription of the VA1 gene in vitro. Furthermore, TFIIIC35 specifically interacts with the human TFIIIC subunits TFIIIC63 and, to a lesser extent, TFIIIC90 in vitro. Finally, we determined a limited region in the smallest subunit of yeast TFIIIC that is sufficient for interacting with
the yeast TFIIIC subunit ScTfc1 (orthologous to TFIIIC63) and found it to be adjacent to and overlap the 34-amino acid domain
that is conserved from yeast to humans.
Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.
Mammalian short interspersed elements (SINEs) are abundant retrotransposons that have long been considered junk DNA; however, RNAs transcribed from mouse B2 and human Alu SINEs have recently been found to control mRNA production at multiple levels. Upon cell stress B2 and Alu RNAs bind RNA polymerase II (Pol II) and repress transcription of some protein-encoding genes. Bi-directional transcription of a B2 SINE establishes a boundary that places the growth hormone locus in a permissive chromatin state during mouse development. Alu RNAs embedded in Pol II transcripts can promote evolution and proteome diversity through exonization via alternative splicing. Given the diverse means by which SINE encoded RNAs impact production of mRNAs, this genomic junk is proving to contain hidden gems.
Gene duplications and their subsequent divergence play an important part in the evolution of novel gene functions. Several models for the emergence, maintenance and evolution of gene copies have been proposed. However, a clear consensus on how gene duplications are fixed and maintained in genomes is lacking. Here, we present a comprehensive classification of the models that are relevant to all stages of the evolution of gene duplications. Each model predicts a unique combination of evolutionary dynamics and functional properties. Setting out these predictions is an important step towards identifying the main mechanisms that are involved in the evolution of gene duplications.
The number of subunits of RNA polymerases (RNAPs) increases during evolution from 5 in eubacteria to 12 in archaea. In eukaryotes,
which have at least three RNAPs, the number of subunits has expanded from 12 in RNA polymerase II (RNAPII) to 14 in RNA polymerase
I (RNAPI) and to 17 in RNA polymerase III (RNAPIII). It was recently demonstrated that the two additional subunits found in
RNAPI relative to RNAPII are homologous to TFIIF, a dimeric general transcription factor of RNAPII. Here, we extend this finding
by demonstrating that four of the five RNAPIII-specific subunits are also homologous to transcription factors of RNAPII. We
use the available evidence to propose an evolutionary history of the eukaryotic RNAPs and argue that the increases in the
number of subunits that occurred in RNAPs I and III are due to the permanent recruitment of preexisting transcription factors.
In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.
Three distinct RNA polymerase activities have been isolated from developing sea urchin embryos. In rat liver nuclei there are two RNA polymerase activities. One polymerase (I) is probably localized in the nucleolus and one (II) in the nucleoplasm.
Most small nuclear RNA (snRNA) genes are transcribed by RNA polymerase II, but some (e.g., U6) are transcribed by RNA polymerase
III. In vertebrates a TATA box at a fixed distance downstream of the proximal sequence element (PSE) acts as a dominant determinant
for recruiting RNA polymerase III to U6 gene promoters. In contrast, vertebrate snRNA genes that contain a PSE but lack a
TATA box are transcribed by RNA polymerase II. In plants, transcription of both classes of snRNA genes requires a TATA box
in addition to an upstream sequence element (USE), and polymerase specificity is determined by the spacing between these two
core promoter elements. In these examples, the PSE (or USE) is interchangeable between the two classes of snRNA genes. Here
we report the surprising finding that the Drosophila U1 and U6 PSEs cannot functionally substitute for each other; rather, determination of RNA polymerase specificity is an intrinsic
property of the PSE sequence itself. The alteration of two or three base pairs near the 3′-end of the U1 and U6 PSEs was sufficient
to switch the RNA polymerase specificity of Drosophila snRNA promoters in vitro. These findings reveal a novel mechanism for achieving RNA polymerase specificity at insect snRNA promoters.
It has been generally accepted that the TATA binding protein (TBP) is a universal mediator of transcription by RNA polymerase I, II, and III. Here we report that the TBP-related factor TRF1 rather than TBP is responsible for RNA polymerase III transcription in Drosophila. Immunoprecipitation and in vitro transcription assays using immunodepleted extracts supplemented with recombinant proteins reveals that a TRF1:BRF complex is required to reconstitute transcription of tRNA, 5S and U6 RNA genes. In vivo, the majority of TRF1 is complexed with BRF and these two proteins colocalize at many polytene chromosome sites containing RNA pol III genes. These data suggest that in Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a major role in RNA pol III transcription.
A key step in retrieving the information stored in the complex genomes of eukaryotes involves the identification of transcription units and, more specifically, the recognition of promoter sequences by RNA polymerase. In eukaryotes, the task of recognizing nuclear gene promoters and then transcribing the genes is divided among three highly related enzymes, RNA polymerases I, II, and III. Each of these RNA polymerases is dedicated to the transcription of specific sets of genes, and each depends on accessory factors, the so-called transcription factors, to recognize its cognate promoter sequences.
Transcription factor IIIB (TFIIIB), consisting of the TATA-binding protein (TBP), TFIIB-related factor (Brf1) and Bdp1, is a central component in basal and regulated transcription by RNA polymerase III. TFIIIB recruits its polymerase to the promoter and subsequently has an essential role in the formation of the open initiation complex. The amino-terminal half of Brf1 shares a high degree of sequence similarity with the polymerase II general transcription factor TFIIB, but it is the carboxy-terminal half of Brf1 that contributes most of its binding affinity with TBP. The principal anchoring region is located between residues 435 and 545 of yeast Brf1, comprising its homology domain II. The same region also provides the primary interface for assembling Bdp1 into the TFIIIB complex. We report here a 2.95 A resolution crystal structure of the ternary complex containing Brf1 homology domain II, the conserved region of TBP and 19 base pairs of U6 promoter DNA. The structure reveals the core interface for assembly of TFIIIB and demonstrates how the loosely packed Brf1 domain achieves remarkable binding specificity with the convex and lateral surfaces of TBP.
This chapter discusses the structure and function of RNA polymerase II. High-resolution structural studies of polymerase II (Pol II) by x-ray crystallography required large amounts of pure protein that cannot be obtained by over-expression because of the complexity of the enzyme. The described structural studies of yeast Pol II are directly relevant to Pol II enzymes in higher organisms, because Pol II subunits are very well conserved in sequence and function. After successful RNA chain elongation, transcription terminates and Pol II dissociates from the template. Some of the steps during the transcription cycle can be carried out by Pol II alone. Pol II can maintain an open transcription bubble, translocate along the template DNA, synthesize RNA from the template, and proofread the nascent RNA. However, for all other steps during the transcription cycle, Pol II requires additional proteins. The chapter presents backbone models of the complete initiation-competent Pol II, including the Rpb4/7 complex and the complete Pol II with bound elongation factor transcription factor IIS (TFIIS). The structures, together with many functional studies, have given many insights into the mechanism of mRNA transcription. Structural and functional studies of bacterial RNA polymerase allow for interesting comparisons and evolutionary considerations. The Pol II structures now guide mutagenesis experiments aimed at a dissection of the transcription mechanism.
Metazoans have evolved multiple paralogues of the TATA binding protein (TBP), adding another tunable level of gene control at core promoters. While TBP-related factor 1 (TRF1) shares extensive homology with TBP and can direct both Pol II and Pol III transcription in vitro, TRF1 target sites in vivo have remained elusive. Here, we report the genome-wide identification of TRF1-binding sites using high-resolution genome tiling microarrays. We found 354 TRF1-binding sites genome-wide with approximately 78% of these sites displaying colocalization with BRF. Strikingly, the majority of TRF1 target genes are Pol III-dependent small noncoding RNAs such as tRNAs and small nonmessenger RNAs. We provide direct evidence that the TRF1/BRF complex is functionally required for the activity of two novel TRF1 targets (7SL RNA and small nucleolar RNAs). Our studies suggest that unlike most other eukaryotic organisms that rely on TBP for Pol III transcription, in Drosophila and possibly other insects the alternative TRF1/BRF complex appears responsible for the initiation of all known classes of Pol III transcription.
The role of RNA polymerase (Pol) III in eukaryotic transcription is commonly thought of as being restricted to a small set of highly expressed, housekeeping non-protein-coding (nc)RNA genes. Recent studies, however, have remarkably expanded the set of known Pol III-synthesized ncRNAs, suggesting that gene-specific Pol III regulation is more common than previously appreciated. Newly identified Pol III transcripts include small nucleolar RNAs, microRNAs, short interspersed nuclear element-encoded or tRNA-derived RNAs and novel classes of ncRNA that can display significant sequence complementarity to protein-coding genes and might thus regulate their expression. The extent of the Pol III transcriptome, the complexity of its regulation and its influence on cell physiology, development and disease are emerging as new areas for future research.