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1.
Nucleic Acids Res ; 52(11): 6220-6233, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38613396

RESUMO

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3'-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3'-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.


Assuntos
Íntrons , Complexo Mediador , Regiões Promotoras Genéticas , Proteínas Ribossômicas , Proteínas de Saccharomyces cerevisiae , Sítios de Ligação , Regulação Fúngica da Expressão Gênica , Íntrons/genética , Complexo Mediador/metabolismo , Complexo Mediador/genética , Ligação Proteica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
Nucleic Acids Res ; 47(9): 4814-4830, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916349

RESUMO

Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure and function of its regulatory factor Kti12 have remained elusive. Here, we present the crystal structure of Kti12's nucleotide hydrolase domain trapped in a transition state of ATP hydrolysis. The structure reveals striking similarities to an O-phosphoseryl-tRNA kinase involved in the selenocysteine pathway. Both proteins employ similar mechanisms of tRNA binding and show tRNASec-dependent ATPase activity. In addition, we demonstrate that Kti12 binds directly to Elongator and that ATP hydrolysis is crucial for Elongator to maintain proper tRNA anticodon modification levels in vivo. In summary, our data reveal a hitherto uncharacterized link between two translational control pathways that regulate selenocysteine incorporation and affect ribosomal tRNA selection via specific tRNA modifications.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Adenosina Trifosfatases/química , Anticódon/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Chaetomium/química , Chaetomium/enzimologia , Cristalografia por Raios X , Conformação Proteica , RNA de Transferência/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Uridina/genética
3.
PLoS Genet ; 11(1): e1004931, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25569479

RESUMO

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.


Assuntos
Caseína Quinase I/genética , Histona Acetiltransferases/genética , Fatores de Alongamento de Peptídeos/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alanina/genética , Caseína Quinase I/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Complexos Multiproteicos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fenótipo , Fosforilação , Proteínas de Saccharomyces cerevisiae/metabolismo , Uridina/genética
4.
Mol Microbiol ; 94(6): 1213-26, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25352115

RESUMO

Diphthamide is a conserved modification in archaeal and eukaryal translation elongation factor 2 (EF2). Its name refers to the target function for diphtheria toxin, the disease-causing agent that, through ADP ribosylation of diphthamide, causes irreversible inactivation of EF2 and cell death. Although this clearly emphasizes a pathobiological role for diphthamide, its physiological function is unclear, and precisely why cells need EF2 to contain diphthamide is hardly understood. Nonetheless, the conservation of diphthamide biosynthesis together with syndromes (i.e. ribosomal frame-shifting, embryonic lethality, neurodegeneration and cancer) typical of mutant cells that cannot make it strongly suggests that diphthamide-modified EF2 occupies an important and translation-related role in cell proliferation and development. Whether this is structural and/or regulatory remains to be seen. However, recent progress in dissecting the diphthamide gene network (DPH1-DPH7) from the budding yeast Saccharomyces cerevisiae has significantly advanced our understanding of the mechanisms required to initiate and complete diphthamide synthesis on EF2. Here, we review recent developments in the field that not only have provided novel, previously overlooked and unexpected insights into the pathway and the biochemical players required for diphthamide synthesis but also are likely to foster innovative studies into the potential regulation of diphthamide, and importantly, its ill-defined biological role.


Assuntos
Histidina/análogos & derivados , Saccharomyces cerevisiae/metabolismo , Evolução Molecular , Histidina/genética , Histidina/metabolismo , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Toxins (Basel) ; 9(9)2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28872616

RESUMO

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin's action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Arabidopsis/genética , Fatores Matadores de Levedura/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Arabidopsis/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Mol Cells ; 38(3): 243-50, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25518926

RESUMO

Patterning of the polar axis during the early leaf developmental stage is established by cell-to-cell communication between the shoot apical meristem (SAM) and the leaf primordia. In a previous study, we showed that the DRL1 gene, which encodes a homolog of the Elongator-associated protein KTI12 of yeast, acts as a positive regulator of adaxial leaf patterning and shoot meristem activity. To determine the evolutionally conserved functions of DRL1, we performed a comparison of the deduced amino acid sequence of DRL1 and its yeast homolog, KTI12, and found that while overall homology was low, well-conserved domains were presented. DRL1 contained two conserved plant-specific domains. Expression of the DRL1 gene in a yeast KTI12-deficient yeast mutant suppressed the growth retardation phenotype, but did not rescue the caffeine sensitivity, indicating that the role of Arabidopsis Elongator-associated protein is partially conserved with yeast KTI12, but may have changed between yeast and plants in response to caffeine during the course of evolution. In addition, elevated expression of DRL1 gene triggered zymocin sensitivity, while overexpression of KTI12 maintained zymocin resistance, indicating that the function of Arabidopsis DRL1 may not overlap with yeast KTI12 with regards to toxin sensitivity. In this study, expression analysis showed that class-I KNOX genes were downregulated in the shoot apex, and that YAB and KAN were upregulated in leaves of the Arabidopsis drl1-101 mutant. Our results provide insight into the communication network between the SAM and leaf primordia required for the establishment of leaf polarity by mediating histone acetylation or through other mechanisms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Proteínas de Ligação ao GTP/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Cafeína/farmacologia , Sequência Conservada , Proteínas de Ligação ao GTP/química , Teste de Complementação Genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Estresse Fisiológico
7.
Toxins (Basel) ; 5(5): 958-68, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23645155

RESUMO

Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin.


Assuntos
Histidina/análogos & derivados , Proteínas de Saccharomyces cerevisiae/genética , Toxina Diftérica/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina/genética , Histidina/metabolismo , Indenos/farmacologia , Mutação , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
J Bacteriol ; 187(15): 5166-78, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16030210

RESUMO

The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-P(S), was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-P(V). E(sigma)E was necessary and sufficient for P(S) expression in vitro. P(S) expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of sigma(E) is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP approximately P) repressed P(S) in vitro via direct binding to the promoter, the first example of an E(sigma)E-responsive promoter that is repressed by PhoP approximately P. Whereas either PhoP or PhoP approximately P in the presence of E(sigma)A was sufficient to stimulate transcription from the phoB-P(V) promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP approximately P were required for P(V) promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P(i)-replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Óperon , Fosforilação , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Esporos Bacterianos/genética , Fatores de Transcrição/genética
9.
J Bacteriol ; 186(5): 1493-502, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14973033

RESUMO

Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP(I206A), PhoP(R210A), PhoP(L209A), and PhoP(H208A)) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP(H205A) and PhoP(V204A)) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP(V202A) and PhoP(D203A), had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP(V190A), PhoP(W191A), PhoP(Y193A), PhoP(F195A), PhoP(G197A,) PhoP(T199A), and PhoP(R200A)) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP(W191A) and PhoP(R200A) could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short alpha helix 3, more similar to OmpR than to PhoB of Escherichia coli.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Ativação Transcricional , Alanina , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Regulação Bacteriana da Expressão Gênica , Isopropiltiogalactosídeo/farmacologia , Mutagênese , Estrutura Secundária de Proteína , Transcrição Gênica
10.
Acta Microbiol Pol ; 51(3): 225-35, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12588097

RESUMO

Statistically designed experimental strategy has been performed in order to evaluate and optimize nutritional and environmental parameters that affect ferrous ion oxidation rate in Acidithiobacillus ferrooxidans ATCC 23270. Plackett-Burman design was carried out to evaluate efficiently the biological significance of 10 culture conditions influencing ferrous-ion oxidation rate of A. ferrooxidans grown for 5 days in shake-flask batch mode on the newly modified 9-K media. Among ten fermentation factors examined, the most significant variables influencing ferrous-ion oxidation rate were statistically elucidated to be pH and calcium nitrate as positive contributors, whereas trace metals solution and potassium chloride were the most significant negative contributors. The optimal levels of the most significant three nutritional factors were further predicted from a polynomial model created from the data obtained from three level factorial design, a Box-Behnken design. Predicted optimal ferrous-ion oxidation rate Q(Fe2+) was recorded to be 0.148 (g Fe2+/l/hr). On verifying the predicted value, an experiment was performed under optimal predicted conditions and showed an actual experimental Q(Fe2+) of 0.152 g/l/hr, which was 2.7% over the predicted value. Our optimized medium formula gave overall five folds increase in ferrous-ion oxidation rates over the previously published data of standard 9-K medium on batch culture of A. ferrooxidans ATCC 23270 with higher mu(max) (hr(-1)) of 0.177 which was achieved within 75 h incubation in shake-flask culture.


Assuntos
Compostos Ferrosos/metabolismo , Modelos Biológicos , Modelos Estatísticos , Thiobacillus/metabolismo , Compostos de Cálcio/metabolismo , Meios de Cultura , Fermentação , Concentração de Íons de Hidrogênio , Nitratos/metabolismo , Oxirredução , Cloreto de Potássio/metabolismo , Oligoelementos/metabolismo
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