Transcription and pre-mRNA processing, e.g., splicing, occur at the same place and time in the context of chromatin. A growing amount of evidence supports the hypothesis that these processes are interconnected. Prp45/SKIP is one of the factors which are believed to mediate the interconnection. The human ortholog, SKIP, is known for affecting mRNA formation on the levels of transcription initiation and elongation. Moreover, it interacts with chromatin modifiers and it is a splicing factor, too. The function of the Saccharomyces cerevisiae ortholog, Prp45, has been so far connected only to pre-mRNA splicing. In this work, we characterized the role of Prp45 in splicing and elaborated the results connecting Prp45 to transcription and chromatin modifications. RNA-seq results showed that pre-mRNA is accumulated in prp45(1-169) cells. This accumulation is not caused by the reduced activity of pathways responsible for RNA degradation. The extent of the splicing inefficiency in prp45(1-169) cells did not depend on either the canonicity of the 5' splice site and branch site or the distance between the branch site and the 3' splice site. Using chromatin immunoprecipitation, we found that prp45(1-169) mutation causes delay in U2 snRNP recruitment to assembling spliceosome. This delay transfers to the later...

Chromobacterium violaceum é uma bactéria de vida livre, móvel, que habita fontes de água e solos pobres de regiões tropicais e subtropicais. Nestes habitats, em que a concentração de fosfato é baixa, o regulon PHO encontra-se ativado. No laboratório, esta bactéria é capaz de crescer razoavelmente bem, apesar da limitação de fosfato, atingindo um rendimento celular similar ao observado na abundância deste nutriente. Mutações nos genes pstS, phoU, phoA1 e phoA2 foram construídas. As mutações pstS e phoU não causaram qualquer alteração no padrão de expressão da fosfatase alcalina, sugerindo que estes genes não participam da repressão dos genes de PHO. Porém, o mutante pstS mostrou-se deficiente na captação de Pi. Os mutantes phoA1 e phoA2 apresentaram, cada um, severa redução na atividade da fosfatase alcalina. Foi investigada a possibilidade de PhoA1 e PhoA2 formarem uma proteína heterodimérica. Fusões transcricionais entre phoU e phoB ao gene lacZ, revelaram que estes genes respondem à carência de Pi. Surpreendentemente, C. violaceum se mostrou pouco resistente a estresses ambientais. / Chromobacterium violaceum is a free-living, mobile bacterium that inhabits water sources and soils of tropical and subtropical regions, where the phosphate concentration is low. The PHO regulon is activated in response to low phosphate concentration in the environment. Surprisingly, the growth yield of C. violaceum under phosphate excess or under phosphate limitation is very similar. Mutations in pstS, phoU, phoA1 and phoA2 genes were constructed. The expression of alklaine phosphatase was not affected by the phoU and pstS mutations, suggesting that these genes do not participate in the repression of the PHO regulon. However, the pstS mutant was deficient in the uptake of Pi. The phoA1 and phoA2 mutants presented each one a severe reduction in alkaline phosphatase activity. The hypothesis that PhoA1 and PhoA2 form a heterodimeric protein was investigated. Transcriptional fusions between the promoters of phoB and phoU to lacZ showed that these genes respond to Pi starvation. Stress resistance assays showed that C. violaceum is generally sensitive to environmental stresses.

Chromobacterium violaceum é uma bactéria de vida livre, móvel, que habita fontes de água e solos pobres de regiões tropicais e subtropicais. Nestes habitats, em que a concentração de fosfato é baixa, o regulon PHO encontra-se ativado. No laboratório, esta bactéria é capaz de crescer razoavelmente bem, apesar da limitação de fosfato, atingindo um rendimento celular similar ao observado na abundância deste nutriente. Mutações nos genes pstS, phoU, phoA1 e phoA2 foram construídas. As mutações pstS e phoU não causaram qualquer alteração no padrão de expressão da fosfatase alcalina, sugerindo que estes genes não participam da repressão dos genes de PHO. Porém, o mutante pstS mostrou-se deficiente na captação de Pi. Os mutantes phoA1 e phoA2 apresentaram, cada um, severa redução na atividade da fosfatase alcalina. Foi investigada a possibilidade de PhoA1 e PhoA2 formarem uma proteína heterodimérica. Fusões transcricionais entre phoU e phoB ao gene lacZ, revelaram que estes genes respondem à carência de Pi. Surpreendentemente, C. violaceum se mostrou pouco resistente a estresses ambientais. / Chromobacterium violaceum is a free-living, mobile bacterium that inhabits water sources and soils of tropical and subtropical regions, where the phosphate concentration is low. The PHO regulon is activated in response to low phosphate concentration in the environment. Surprisingly, the growth yield of C. violaceum under phosphate excess or under phosphate limitation is very similar. Mutations in pstS, phoU, phoA1 and phoA2 genes were constructed. The expression of alklaine phosphatase was not affected by the phoU and pstS mutations, suggesting that these genes do not participate in the repression of the PHO regulon. However, the pstS mutant was deficient in the uptake of Pi. The phoA1 and phoA2 mutants presented each one a severe reduction in alkaline phosphatase activity. The hypothesis that PhoA1 and PhoA2 form a heterodimeric protein was investigated. Transcriptional fusions between the promoters of phoB and phoU to lacZ showed that these genes respond to Pi starvation. Stress resistance assays showed that C. violaceum is generally sensitive to environmental stresses.

The Pho regulon is controlled by the PstSCAB transporter, PhoU, and the two-component proteins, PhoB and PhoR. PhoU is a negative regulator of the Pho regulon under phosphate-replete conditions. How PhoU functions is unknown. Many PhoU homologues are found widely throughout prokaryotic domains. There are several conserved amino acid residues in the PhoU protein. It is hypothesized that these residues play an important role in the function of PhoU. To test this hypothesis, several site directed mutations in the phoU gene have been produced with single amino acid changes in conserved residues. After testing these mutants, it was found that some of the mutants abolished repression of the Pho regulon while other mutants had little or no effect. Further study of these mutants and their phenotypes will reveal more about how PhoU functions and help to better understand bacterial signaling in general.

No description available.

O operon pst de P. aeruginosa codifica um transportador de fosfato de alta afinida-de e também a proteína PhoU que, em conjunto, atuam como repressores da ex-pressão do regulon Pho dessa espécie. A atividade de PhoU está também associada ao metabolismo de polifosfato (poliP), dado que mutantes phoU nulos apresentam um vasto acúmulo do biopolímero. Ensaios de β-galactosidase mostraram uma alteração na expressão dos genes ppk e ppx, envolvidos no metabolismo de poliP, no mutante phoU. Observou-se que na cepa selvagem, a transcrição de ppk e de ppx não responde às limitações de Pi ou de nitrogênio, sendo esses genes altamente expressos em condições normais de crescimento. Além disso, determinou-se que ppk é co-transcrito com o gene hemB, os quais formam, portanto, um operon. O operon pst também foi analisado. Foi identificado por ensaios de northern blot o transcrito do primeiro gene do operon, pstS, que codifica uma proteína periplasmática. Também, foi identificado um promotor imediatamente a montante de phoU, o gene mais distal do operon, que permitiria sua expressão em condições normais do crescimento bacteriano. Por fim, determinou-se por ensaios de EMSA que as duas sequências consenso Pho box presentes no operon pst são completamente funcionais. / The pst operon in P. aeruginosa encodes a high-affinity phosphate transporter and the PhoU protein, which together act as repressors of Pho regulon of this species. The PhoU activity is also related with polyphosphate (polyP) metabolism, since phoU null mutants have a large accumulation of the biopolymer. β-galactosidase assays allowed to confirm a change in the expression of ppk and ppx genes, in-volved in PolyP metabolism, in the phoU mutant. It was also evidenced that in the wild type strain, the ppk and ppx transcription does not respond to Pi or nitrogen starvation, and that these genes are highly expressed under conditions of normal growth. In addition, it was determined that ppk is co-transcribed with hemB, a gene involved in the synthesis of porphyrins, and they constitute therefore an operon. The pst operon was also examined. Was identified by northern blot the transcript of the first gene in the operon, pstS, which encodes a periplasmic protein. Also, a promoter was identified immediately upstream of phoU, the most distal gene in the operon, allowing its expression in normal conditions of bacterial growth. Finally, it was determined by EMSA that the two consensus sequences Pho box present in the pst operon are fully functional.

Inorganic phosphate is an essential nutrient required by all organisms for optimal growth. During phosphate starvation, Saccharomyces cerevisiae induces a set of genes responsible for the regulation of inorganic phosphate acquisition. The phosphate-responsive signaling (PHO) pathway controls this response, with the CDK-cyclin complex Pho85-Pho80 playing a prominent role. Here we report the X-ray structure of the Pho85-Pho80 complex, identifying the unique structural features that distinguish it from other cell cycle associated CDK-cyclin complexes. The structure reveals a specific salt bridge between a Pho85 arginine and a Pho80 aspartate that maintains a Pho80 loop confirmation important for substrate recognition and makes phosphorylation of the Pho85 activation loop dispensible. We show that a cluster of residues distal to the kinase active site are involved in a high affinity interaction between the Pho80 cyclin and the transcription factor substrate (Pho4). The structure also reveals a separate high affinity binding site for the CDK inhibitor (Pho81). The fission yeast, Schizosaccharomyces pombe, regulates expression of the secreted acid phosphatase \((pho1^+)\) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive \((pho7^+)\) and negative \((csk1^+)\) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq), and global transcriptome sequencing (RNA-Seq) to characterize the role of \(pho7^+\) and \(csk1^+\) in the PHO response. We show that there is a fast and slow response to phosphate starvation, each with defined regulatory roles. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding dynamics in response to phosphate-limitation and Csk1 activity. We identify a conserved PHO response for the PHO5 \((pho1^+)\), PHO84 \((spbc8e4.01c^+)\), and GIT1 \((spbc1271.09^+)\) orthologs. We show that activation of \(pho1^+\) requires Pho7 binding to a UAS in the \(pho1^+\) promoter and that a URS is necessary for Csk1 repression. We find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require \(pho7^+\) for maximal induction. Using RNA-Seq we show that Pho7 is also involved in regulating non-coding transcription and is a bi-functional transcription factor.

O operon pst de P. aeruginosa codifica um transportador de fosfato de alta afinida-de e também a proteína PhoU que, em conjunto, atuam como repressores da ex-pressão do regulon Pho dessa espécie. A atividade de PhoU está também associada ao metabolismo de polifosfato (poliP), dado que mutantes phoU nulos apresentam um vasto acúmulo do biopolímero. Ensaios de β-galactosidase mostraram uma alteração na expressão dos genes ppk e ppx, envolvidos no metabolismo de poliP, no mutante phoU. Observou-se que na cepa selvagem, a transcrição de ppk e de ppx não responde às limitações de Pi ou de nitrogênio, sendo esses genes altamente expressos em condições normais de crescimento. Além disso, determinou-se que ppk é co-transcrito com o gene hemB, os quais formam, portanto, um operon. O operon pst também foi analisado. Foi identificado por ensaios de northern blot o transcrito do primeiro gene do operon, pstS, que codifica uma proteína periplasmática. Também, foi identificado um promotor imediatamente a montante de phoU, o gene mais distal do operon, que permitiria sua expressão em condições normais do crescimento bacteriano. Por fim, determinou-se por ensaios de EMSA que as duas sequências consenso Pho box presentes no operon pst são completamente funcionais. / The pst operon in P. aeruginosa encodes a high-affinity phosphate transporter and the PhoU protein, which together act as repressors of Pho regulon of this species. The PhoU activity is also related with polyphosphate (polyP) metabolism, since phoU null mutants have a large accumulation of the biopolymer. β-galactosidase assays allowed to confirm a change in the expression of ppk and ppx genes, in-volved in PolyP metabolism, in the phoU mutant. It was also evidenced that in the wild type strain, the ppk and ppx transcription does not respond to Pi or nitrogen starvation, and that these genes are highly expressed under conditions of normal growth. In addition, it was determined that ppk is co-transcribed with hemB, a gene involved in the synthesis of porphyrins, and they constitute therefore an operon. The pst operon was also examined. Was identified by northern blot the transcript of the first gene in the operon, pstS, which encodes a periplasmic protein. Also, a promoter was identified immediately upstream of phoU, the most distal gene in the operon, allowing its expression in normal conditions of bacterial growth. Finally, it was determined by EMSA that the two consensus sequences Pho box present in the pst operon are fully functional.

The PstSCAB2 complex serves the dual function of being a phosphate transporter as well as the primary sensor of phosphate for the Pho regulon. PhoU is an integral protein required for the signal from PstSCAB2 to be transmitted to PhoR. Our hypothesis is that conformational changes of PstSCAB2 during the phosphate transport process are the mechanism by which information about environmental phosphate levels are transduced to the cell. Additionally, we propose that direct protein-protein interactions between PhoU and the alternating conformations of PstSCAB2 mediate PhoU interactions with PhoR. By means of genetic and biochemical approaches, we have found substantial evidence supporting both these hypotheses.

A habilidade de sensoriar o ambiente extracelular e responder às suas mudanças é inerente para a maioria das bactérias. As concentrações de nutrientes direcionam os processos metabólicos relacionados à sobrevivência e proliferação. O fosfato inorgânico (Pi) é um dos nutrientes cuja regulação, sensoriamento e sinalização são bastante conservados em bactéria. Um dos mecanismos de captação do íon fosfato com alta afinidade é o sistema Pst, um transportador do tipo ABC (ATP-Binding Cassette) , localizado na membrana interna das células. Este transportador, juntamente com as proteínas PhoR/PhoB que formam um sistema de dois componentes (Two-Component Regulatory System) , são capazes de sensoriar e monitorar os níveis deste íon nas células. Ambos os sistemas pertencem ao chamado regulon Pho, conjunto de genes envolvidos no transporte, captação e metabolização do fosfato. Estudos tem mostrado que a interação entre os sistema Pst e o sistema doiscomponentes PhoR/PhoB é mediada pela proteína PhoU, um regulador negativo cujo gene encontra-se no mesmo operon do transportador. Apesar de muito estudados em Escherichia coli , poucas informações existem sobre as características destes sistemas em Xanthomonas citri subsp. citri , bactéria responsável pelo cancro cítrico e de grande importância econômica para o país. Estudos realizados pelo nosso grupo mostraram que X. citri conserva a maioria dos genes descritos como pertencentes ao regulon Pho, incluindo o sistema Pst, as proteínas PhoR/PhoB e PhoU. Este trabalho, portanto, tem como objetivos, a caracterização funcional e estrutural da proteína PhoU de X. citri e a análise da possível interação de PhoU com a proteína PhoR, a histidina quinase do sistema dois componentes. Para tal, as proteínas foram expressas em linhagens de E. coli Tuner e purificadas por cromatografia de afinidade a metal, seguida de exclusão molecular. Visando a caracterização biofísica e estrutural da proteína PhoU, foram realizados ensaios de dicroísmo circular, cristalização, análises de bioinformática e modelagem molecular. Os resultados de bioinformática mostraram que PhoU conserva características estruturais e funcionais quando comparada com ortólogos. Após sua purificação, a proteína foi produzida na sua forma enovelada e mostrou interação com ligantes, conforme descrito na literatura para ortólogos. A expressão da proteína PhoR também foi obtida e ensaios de pull-down foram realizados para a caracterização da interação entre PhoU-PhoR. Adicionalmente, foram realizados estudos de expressão das proteínas em diferentes condições de cultivo, utilizando-se anticorpos policlonais anti-PhoU e anti-PhoR. Os resultados apresentados neste projeto são de grande importância uma vez que se obteve a padronização dos processos de produção de ambas as proteínas e ensaios biofísicos e estruturais para a futura caracterização do complexo, o que será de grande relevância para a compreensão do papel destes sistemas na fisiologia da bactéria. / The ability to sensor the extracellular environment and respond to its changes is inherent to most bacteria. Nutrient concentrations direct metabolic processes related to survival and proliferation. Inorganic phosphate (Pi) is one of the nutrients whose regulation, sensing and signalling are quite preserved in bacteria. One of the mechanisms for phosphate ion uptake with high affinity is the Pst system, composed by an ABC transporter (ATP-Binding Cassette), located on the inner membrane of the cells. This transporter, along with the PhoR/PhoB proteins, which form a Two Component Regulatory System, are capable of sensing and monitoring the levels of phosphate in cells. Both systems belong to the called regulon Pho, set of genes involved in phosphate transport, uptake and metabolism. Studies have shown that the interaction between the Pst system and the two component PhoR/PhoB system is mediated by the PhoU protein, a negative regulator, whose gene is located in the same operon of Pst system. Although much studied in Escherichia coli , there are few information about of these systems in Xanthomonas citri subsp. citri , the major causative of citric canker. Studies conducted by our group showed that X. citri conserves most of the genes described as belonging to regulon Pho, including the Pst system, the proteins PhoR/PhoB and PhoU. This work, therefore, aimed at performing functional and structural characterization of the X. citri PhoU protein and analyzing the possible interaction of PhoU with the PhoR protein, the histidine kinase of the Two Component System. For this, the proteins were expressed in E. coli Tuner strains and purified by metal affinity chromatography, followed by size exclusion chromatography. Aiming at the biophysical and structural characterization of the PhoU protein, we performed circular dichroism, crystallization, bioinformatics and molecular modeling. The results of bioinformatics showed that PhoU retains structural and functional characteristics when compared with orthologs. After purification, the protein was produced in its folded form and showed interaction with ligands, as described in the literature for orthologs. Expression of the PhoR protein was also obtained and Pull Down assays were performed for the characterization of the interaction between PhoUPhoR. In addition, protein expression studies were carried out under different culture conditions using polyclonal anti-PhoU and anti-PhoR antibodies. The results presented in this project are of great importance, once the standardization of the production processes of both proteins has been obtained, as well as biophysical and structural information. These information will be important for future characterization of the complex, which will be of great relevance for the understanding of the role of these systems in the physiology of bacteria.