Eukaryotic Class Research Articles

Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase

Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K(m) value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.

Inhibition of RNA Polymerase III Elongation by a T10 Peptide Nucleic Acid

The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs). We analyzed the effect of a T10 PNA on in vitro transcription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues. At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner. The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription. Thus, an impairment of pol III recycling fully accounts for the observed inhibition. As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA adduct before reaching the terminator region. On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs. Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription.

Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G ( rdn15) and U1495C ( hyg1) in helix 44 of the decoding region and by U912C ( rdn4) and G886A ( rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI +] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI +] -mediated stop codon read-through but do not cure cells of the [PSI +] prion. Interestingly, cells bearing hyg1 seem to enable [PSI +] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI +] , is partly relieved.

DNA Bends in TATA-binding Protein·TATA Complexes in Solution Are DNA Sequence-dependent

The TATA-binding protein (TBP) initiates assembly of transcription preinitiation complexes on eukaryotic class II promoters, binding to and restructuring consensus and variant "TATA box" sequences. The sequence dependence of the DNA structure in TBP-TATA complexes has been investigated in solution using fluorescence resonance energy transfer. The mean 5'dye-3'dye distance varies significantly among oligomers bearing the adenovirus major late promoter sequence (AdMLP) and five single-site variants bound to Saccharomyces cerevisiae TBP, consistent with solution bend angles for AdMLP of 76 degrees and for the variants ranging from 30 degrees to 62 degrees. These solution bends contrast sharply with the corresponding co-crystal structures, which show approximately 80 degrees bends for all sequences. Transcription activities for these TATA sequences are strongly correlated with the solution bend angles but not with TBP-DNA binding affinities. Our results support a model in which transcription efficiency derives primarily from the sequence-dependent structure of the TBP-TATA binary complex. Specifically, the distance distribution for the average solution structure of the TBP-TATA complex may reflect the sequence-dependent probability for the complex to assume a conformation in which the TATA box DNA is severely bent. Upon assumption of this geometry, the binary complex becomes a target for binding and correctly orienting the other components of the preinitiation complex.

DNA Sequence-dependent Differences in TATA-binding Protein-induced DNA Bending in Solution Are Highly Sensitive to Osmolytes

The complex formed between the TATA-binding protein (TBP) and the "TATA box" of eukaryotic class II promoters is the foundation for assembly of the complex to which RNA polymerase II is ultimately recruited. TBP binds productively to canonical and diverse variant TATA sequences with >100-fold differences in transcription efficiency. Co-crystals of canonical sequences and >11 variant sequences bound to various TBP molecules all have approximately 80 degrees bends. In contrast, the bend angles for TBP-TATA complexes in solution, derived from distance distributions, are approximately 80 degrees for a canonical sequence but range from 30 degrees to 62 degrees for five variant sequences. We show in this study that the osmolytes used to crystallize TBP-TATA complexes induce profound increases in the DNA bends of two transcriptionally active TBP-bound variant sequences to a common angle of approximately 80 degrees but have little effect on a transcriptionally inactive variant. The effect of osmolyte on the TBP-induced DNA bend of a variant TATA box sequence is also manifest in the kinetics of association, demonstrating a functional consequence of an osmolyte-induced structural change.

Common phylogeny of catalase-peroxidases and ascorbate peroxidases

Catalase-peroxidases belong to Class I of the plant, fungal, bacterial peroxidase superfamily, together with yeast cytochrome c peroxidase and ascorbate peroxidases. Obviously these bifunctional enzymes arose via gene duplication of an ancestral hydroperoxidase. A 230-residues long homologous region exists in all eukaryotic members of Class I, which is present twice in both prokaryotic and archaeal catalase-peroxidases. The overall structure of eukaryotic Class I peroxidases may be retained in both halves of catalase-peroxidases, with major insertions in several loops, some of which may participate in inter-domain or inter-subunit interactions. Interspecies distances in unrooted phylogenetic trees, analysis of sequence similarities in distinct structural regions, as well as hydrophobic cluster analysis (HCA) suggest that one single tandem duplication had already occurred in the common ancestor prior to the segregation of the archaeal and eubacterial lines. The C-terminal halves of extant catalase-peroxidases clearly did not accumulate random changes, so prolonged periods of independent evolution of the duplicates can be ruled out. Fusion of both copies must have occurred still very early or even in the course of the duplication. We suggest that the sparse representatives of eukaryotic catalase-peroxidases go back to lateral gene transfer, and that, except for several fungi, only single copy hydroperoxidases occur in the eukaryotic lineage. The N-terminal halves of catalase-peroxidases, which reveal higher homology with the single-copy members of the superfamily, obviously are catalytically active, whereas the C-terminal halves of the bifunctional enzymes presumably control the access to the haem pocket and facilitate stable folding. The bifunctional nature of catalase-peroxidases can be ascribed to several unique sequence peculiarities conserved among all N-terminal halves, which most likely will affect the properties of both haem ligands.

A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding

Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.

A Conserved Negatively Charged Amino Acid Modulates Function in Human Nonmuscle Myosin IIA

A myosin surface loop (amino acids 391-404) is postulated to be an important actin binding site. In human beta-cardiac myosin, mutation of arginine-403 to a glutamine or a tryptophan causes hypertrophic cardiomyopathy. There is a phosphorylatable serine or threonine residue present on this loop in some lower eukaryotic myosin class I and myosin class VI molecules. Phosphorylation of the myosin I molecules at this site regulates their enzymatic activity. In almost all other myosins, the homologous residue is either a glutamine or an aspartate, suggesting that a negative charge at this location is important for activity. To study the function of this loop, we have used site-directed mutagenesis and baculovirus expression of a heavy meromyosin- (HMM-) like fragment of human nonmuscle myosin IIA. An R393Q mutation (equivalent to the R403Q mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase or in vitro motility of the expressed HMM-like fragment. Three mutations, D399K, D399A, and a deletion mutation that removes residues 393-402, all decrease both the V(max) of the actin-activated MgATPase by 8-10-fold and the rate of in vitro motility by a factor of 2-3. The K(ATPase) of the actin-activated MgATPase activity and the affinity constant for binding of HMM to actin in the presence of ADP are affected by less than a factor of 2. These data support an important role for the negative charge at this location but show that it is not critical to enzymatic activity.

In VivoStructure–Function Analyses ofCaenorhabditis elegansMEC-4, a Candidate Mechanosensory Ion Channel Subunit

Mechanosensory signaling mediated by mechanically gated ion channels constitutes the basis for the senses of touch and hearing and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. We report a genetically based structure-function analysis of the candidate mechanotransducing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. We identify molecular lesions in 40 EMS-induced mec-4 alleles and further probe residue and domain function using site-directed approaches. Our analysis highlights residues and subdomains critical for MEC-4 activity and suggests possible roles of these in channel assembly and/or function. We describe a class of substitutions that disrupt normal channel activity in touch transduction but remain permissive for neurotoxic channel hyperactivation, and we show that expression of an N-terminal MEC-4 fragment interferes with in vivo channel function. These data advance working models for the MEC-4 mechanotransducing channel and identify residues, unique to MEC-4 or the MEC-4 degenerin subfamily, that might be specifically required for mechanotransducing function. Because many other substitutions identified by our study affect residues conserved within the DEG/ENaC channel superfamily, this work also provides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large number of eukaryotic channel classes, we suggest that similar genetic-based structure-activity studies might be generally applied to generate insight into the in vivo function of diverse channel types.

Fungal transposons: from mobile elements towards molecular tools

Over the last few years an increasing number of transposons has been identified and characterized in filamentous fungi. This includes members of all known eukaryotic classes of transposable elements. Most interestingly, transposons have also been identified in fungi that are used in biotechnology which could provide new tools for strain improvement or gene tagging. Transposons have already been used for diagnostic and population analysis of fungal strains. The first attempts towards transposon-aided gene tagging have been promising and future efforts will almost certainly add a new and powerful method for gene identification in filamentous fungi.

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex

Binding of the TATA-binding protein (TBP) to the “TATA” sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

Splice Mutations in KVLQT1 ?

HomeCirculationVol. 99, No. 18Splice Mutations in KVLQT1? Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBSplice Mutations in KVLQT1? Jessica Tyson, Sue Malcolm and Maria Bitner-Glindzicz Jessica TysonJessica Tyson Molecular Genetics Unit, Institute of Child Health, London, UK Search for more papers by this author , Sue MalcolmSue Malcolm Molecular Genetics Unit, Institute of Child Health, London, UK Search for more papers by this author and Maria Bitner-GlindziczMaria Bitner-Glindzicz Molecular Genetics Unit, Institute of Child Health, London, UK Search for more papers by this author Originally published11 May 1999https://doi.org/10.1161/circ.99.18.2476/aCirculation. 1999;99:2476a–2479To The Editor:Li et al1 propose that 2 new mutations in KVLQT1 in Romano-Ward syndrome (RWS) disrupt splicing, leading to frameshift and premature protein truncation, acting through a loss-of-function mechanism. This is surprising because missense mutations in KVLQT1, acting in a dominant-negative manner, predominate in RWS. In Table 2 of the article by Li et al, 24 of the 26 mutations are missense (the 2 remaining being discussed here). We suggest alternative interpretations.The SP/A249/g-a mutation is a substitution G to A at the third position of codon 249, in which GCG is altered to GCA, occurring at the donor splice junction of exon 6. Mutation of splice sites can cause skipping of the affected exon, activation of cryptic splice sites, or retention of the intron.2 Exon skipping is common. If this occurs in SP/A249/g-a, the predicted protein will lack part of the pore and S6 transmembrane domain but may still multimerise, because if exon 6 is skipped, the resulting mRNA would still read “in frame.” In-frame but deleted transcripts may be stable and translated into protein, as shown for the dystrophin gene.3 The effect of the mutation should be confirmed in RNA, because frameshift and premature truncation of the KVLQT1 protein, as suggested by Li et al, would be a truly novel type of KVLQT1 mutation in RWS.The second mutation is the 3-bp deletion across an exon/intron boundary (denoted SP/V212/ΔGGT), disrupting the 5′ donor site of exon 5. A donor consensus splice site consists of CAG.gtaagt.4 In the patient in the study by Li et al, the wild-type exonic sequence GGG.GTG.gtaagt is mutated to GGG.GTaagt by deletion of 3 nucleotides, Ggt. The authors propose that the mutation SP/V212/ΔGGT alters splicing, leading to a 1-bp deletion in the coding region, causing frameshift and premature truncation of the predicted protein (see Figure ).However, a potential splice sequence, GGG.gtaagt, remains.4 Splicing here would delete a single valine,5 causing a mutant protein capable of dominant-negative action.Missense mutations in KVLQT1 predominate in RWS, whereas nonsense and frameshift mutations predominate in Jervell and Lange-Nielsen syndrome (Tyson, 1998, unpublished data). Carriers of nonsense mutations rarely manifest RWS clinically. The gene product of the null allele should not interfere with the wild-type allele in a dominant-negative manner.Download figureDownload PowerPoint References 1 Li H, Chen Q, Moss AJ, Robinson J, Goytia V, Perry JC, Vincent GM, Priori SG, Lehmann MH, Denfield SW, Duff D, Kaine S, Shimizu W, Schwartz PJ, Wang Q, Towbin JA. New mutations in the KVLQT1 potassium channel that cause long-QT syndrome. Circulation.1998; 97:1264–1269.CrossrefMedlineGoogle Scholar2 Maquat LE. Defects in RNA splicing and the consequence of shortened translational reading frames. Am J Hum Genet.1996; 59:279–286.MedlineGoogle Scholar3 Dunckley MG, Manoharan M, Villiet P, Eperon IC, Dickson G. Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet.1998; 7:1083–1090.CrossrefMedlineGoogle Scholar4 Shapiro M, Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res.1987; 15:7155–7174.CrossrefMedlineGoogle Scholar5 Chouabe C, Neyroud N, Guicheney P, Lazdunski M, Romey G, Barhanin J. Properties of KvLQT1 K+ channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias. EMBO J.1997; 16:5472–5479.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails May 11, 1999Vol 99, Issue 18 Advertisement Article InformationMetrics Copyright © 1999 by American Heart Associationhttps://doi.org/10.1161/circ.99.18.2476/aPMID: 10318740 Originally publishedMay 11, 1999 PDF download Advertisement

Characterization, cloning, and evolutionary history of the chloroplast and cytosolic class I aldolases of the red alga Galdieria sulphuraria.

Two fructose-1,6-bisphosphate aldolases from the acido- and thermophilic red alga Galdieria sulphuraria were purified to apparent homogeneity and N-terminally microsequenced. Both aldolases had similar biochemical properties such as Km (FBP) (5.6-5.8 microM) and molecular masses of the native enzymes (165kDa) as determined by size exclusion chromatography. The subunit size of the purified aldolases, as determined by SDS-PAGE, was 42kDa for both aldolases. The isoenzymes were not inhibited by EDTA or affected by cysteine or potassium ions, implying that they belong to the class I group of aldolases, while other red algae are known to have one class I and one class II aldolase inhibited by EDTA. cDNA clones of the cytosolic and plastidic aldolases were isolated and sequenced. The gene for the cytosolic isoenzyme contained a 303bp untranslated leader sequence, while the gene for the plastidic isoenzyme exhibited a transit sequence of 56 amino-acid residues. Both isoenzymes showed about 48% homology in the deduced amino-acid sequences. A gene tree relates both aldolases to the basis of early eukaryotic class I aldolases. The phylogenetic relationship to other aldolases, particularly to cyanobacterial class II aldolases, is discussed.

New insights into the phylogeny of eukaryotes based on ciliate Hsp70 sequences.

The current framework of the eukaryotic phylogeny is based on the analysis of a comprehensive set of sequences of the small subunit ribosomal RNA. However, phylogenies based on protein-encoding genes are not completely congruent with this picture. Since congruence between different markers is the best tool to determine evolutionary history, we focused on Hsp70 (heat-shock protein of 70 kDa), a chaperone protein which is highly conserved and is a potentially reliable phylogenetic marker. We used a PCR-based approach to sequence Hsp70s in two distinct classes of Ciliates. Seven Hsp70s were identified from Paramecium tetraurelia (Oligohymenophora) and six Hsp70s from Euplotes aediculatus (Hypotricha), encompassing orthologous genes for all major Hsp70 classes of Eukaryotes, i.e., those localized in cytosol, in endoplasmic reticulum, and in mitochondria. Three independent phylogenies of eukaryotes, based on each set of orthologous genes, have been constructed using different tree reconstruction methods. A significant advantage of Hsp70s is the existence of outgroups close to Eukaryotes for these major classes, reducing the long-branch attraction artifact due to the outgroup. The monophyly of Ciliates is supported by good bootstrap proportions in the phylogenetic reconstructions, and this phylum is generally a sister-group of Sporozoa, forming the expected Alveolates clade. The Hsp70 seems to be a suitable phylogenetic marker since it recovers all the monophyletic groups, undoubtedly defined by morphological criteria. The Hsp70 trees are, however, notably different from the rRNA ones and do not show two aspects of the classical topology, i.e., the successive emergence of deeply branching groups and the vast assembly of the major eukaryotic groups, emerging at the tip of the tree, i.e., the "terminal crown". More precisely, the Hsp70 trees do not resolve the relationships between the major groups of Eukaryotes with confidence, in keeping with the hypothesis that all these groups emerged in a great radiation that occurred at the origin of all the extant Eukaryotes.

Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

In vitro metabolism of inositol 1,4,5-trisphosphate by Neurospora crassa

The in vitro metabolism of Ins(1,4,5)P 3 by a soluble and a particulate fraction of Neurospora crassa revealed the presence of an Ins(1,4,5)P 3 kinase activity which was associated with the soluble fraction of this organism but was not detected in the particulate fraction. The product was tentatively identified as Ins(1,3,4,5)P 4 by co-chromatography with an authentic standard. A second minor unidentified InsP 4 was also detected. The kinase was not stimulated by Ca 2+/calmodulin and was unaffected by EGTA. Phosphomonoesterase activity was predominantly associated with the particulate fraction producing Ins(1,4)P 2, Ins(1,5)P 2 and Ins(4,5)P 2 indicating the presence of 1′, 4′ and 5′- phosphomonoesterases. N. crassa therefore differs from other classes of eukaryotes where Ins(1,4,5)P 3 metabolism has been studied in possessing a specific 4′-phosphomonoesterase. In the particulate fraction, 1′-phosphomonoesterase was the principal route for Ins(1,4,5)P 3 degradation whereas all three phosphomonoesterases were equally active in the soluble fraction. All phosphomonoesterase activity was Mg 2+-dependent. Further degradation to inositol monophosphates was not observed under the conditions used in this study.

Considerations of transcriptional control mechanisms: do TFIID-core promoter complexes recapitulate nucleosome-like functions?

The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities. Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins. These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter. Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID. We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions. Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

Analysis of Open Complex Formation during RNA Polymerase II Transcription Initiation Using Heteroduplex Templates and Potassium Permanganate Probing

Open complex formation precedes initiation of transcription by RNA polymerases. In the analysis of transcription initiation from eukaryotic class II promoters, we have used promoter DNA structures that represent intermediates in open complex formation. We describe the preparation and isolation of heteroduplex promoter fragments. Probes containing these DNA structures have a general application in the study of proteins binding to junctions of double- and single-stranded DNA. Such proteins play important roles not only in the regulation of RNA synthesis but also in processes like repair, replication, and recombination of DNA. In addition, a protocol is provided for a rapid and quantitative assay for open complexes and transcription bubbles using potassium permanganate as a chemical probe for single-stranded regions in DNA.

Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo.

Activation of eukaryotic class II gene expression involves the formation of a transcription initiation complex that includes RNA polymerase II, general transcription factors, and SRB components of the holoenzyme. Negative regulators of transcription have been described, but it is not clear whether any are general repressors of class II genes in vivo. We reasoned that defects in truly global negative regulators should compensate for deficiencies in SRB4 because SRB4 plays a positive role in holoenzyme function. Genetic experiments reveal that this is indeed the case: a defect in the yeast homologue of the human negative regulator NC2 (Dr1 x DRAP1) suppresses a mutation in SRB4. Global defects in mRNA synthesis caused by the defective yeast holoenzyme are alleviated by the NC2 suppressing mutation in vivo, indicating that yeast NC2 is a global negative regulator of class II transcription. These results imply that relief from repression at class II promoters is a general feature of gene activation in vivo.

The RNA Subunit of Ribonuclease P from the Zebrafish, Danio rerio

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.