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11	Biochemical, Biophysical and Evolutionary Perspectives of Zinc Finger Proteins in Mycobacterium smegmatis Ghosh, Subho January 2017 (has links) (PDF) Transcription is a major step in expression of genes of a given organism. Due to environmental constrains this step must be regulated in the favour of the sustenance and growth of the organism. Here comes the relevance of transcription factors, mostly proteins which regulate transcription. One such important group of transcription factors is the zinc finger proteins. It is well known that in eukaryotes the C2H2 zinc finger domain containing proteins are the largest group of transcription factors while in prokaryotes the largest group of transcription factors are represented by helix-turn-helix motif containing proteins. Till now only two C2H2 zinc finger domain proteins-Ros and Muc have been found in alpha proteobacteria which are also transcription factors. In eukaryotes the second largest group of zinc finger proteins have their zinc ion coordinated by four cysteine residues- the C4 zinc finger proteins. They make the nuclear hormone receptor superfamily of proteins. They have also been shown to act as transcription factors. But in eubacteria no such proteins have been described in details except an isolated report of crystal structure of a C-terminal zinc finger domain protein- Jann_2411 from Jannaschia sp. Though a lot of transcription factors have been described in mechanistic details in Escherichia coli and Bacillus subtilis, the list of well described mycobacterial transcription factors is short. Given this fact and the lack of any known zinc finger domain transcription factor in actinobacteria we wanted to see whether M. smegmatis genome also encode any homologue of Jann_2411 and if does whether they have ability to modulate transcription. To meet our aim we did BLASTP search against the genome of M. smegmatis using Jann_2411 as query. We found four C-terminal zinc finger domain proteins –Msmeg_0118. Msmeg_3613, Msmeg_3408 and Msmeg_1531, which we named as Mycobacterial single zinc finger protein (Mszfp) and numbered- Mszfp1, Mszfp2, Mszfp3 and Mszfp4, respectively. Mszfp1 and Mszfp2 were chosen for study as they were the top most hits. In this thesis:- Chapter1 introduces zinc finger proteins, transcription and several levels of control of transcription process in eubacteria. In chapter2 we characterised Mszfp1 biophysically and probed its secondary structure content and oligomeric state in the native and demetallated conditions. We have also shown that this conserved hypothetical protein is expressed throughout the growth phase of M. smegmatis, regulated by SigA and SigB. We have also showed that Mszfp1 is a DNA binding protein in the native state and the demetallated protein has altered DNA binding ability. It was noted that on over expression Mszfp1 affects colony morphology and biofilm forming ability, of M. smegmatis. In chapter3 the ability of Mszfp1 to bind to RNA polymerase of M. smegmatis has been explored. It was found that Mszfp1 can activate transcription by interacting with CTD/NTD of α subunit and domain 4 of σA like CRP on type II CRP activated promoter. In chapter4 similar to Mszfp1 the biophysical study of Mszfp2 has been carried out. It was found that Mszfp2 is also a predominantly alpha helical protein with oligomeric structure having DNA binding ability. Similar to Mszfp1 Mszfp2 on over expression changes the colony morphology. Chapter5 deals with the RNA polymerase binding ability of Mszfp2 and its ability to activate transcription by interacting with CTD/NTD of α subunit but not the σA. In chapter6 we have presented a glimpse of the possible biophysical properties of Mszfp3 and Mszfp4 and given a snapshot of distribution of homologues of Mszfps among other actinobacteria. We have also put forward a hypothesis about the origin of C4 and CCHC zinc finger domains. Chapter7 is the summary of the work embedded in the earlier chapters. In Appendix I is described the making of a bacteria (Bacillus licheniformis) driven heat engine. Appendix II describes an effort to study the visco-elastic properties of Mycobacterium smegmatis cells. RNA Polymerase (RNAP) Mszfp2 Mycobacteria Mycobacterium Smegmatis Zinc Finger Protein Mszfp1 Molecular Biophysics
12	Transcriptional Regulation By A Biotin Starvation- And Methanol-Inducible Zinc Finger Protein In The Methylotrophic Yeast, Pichia Pastoris Nallani, Vijay Kumar 11 1900 (has links) (PDF) Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion. Methylotrophic Yeasts Methanol-Inducible Zinc Finger Protein Methanol Metabolism - Gene Repression Pichia pastoris Yeasts - Biotin Biotin Biosynthesis Zinc Finger Protein Mycology
13	Mecanismes de regulació en l'activitat biològica del factor de transcripció Snail Domínguez Solà, David 03 April 2003 (has links) Els factors de transcripció de la família Snail són fonamentals en la "transició epiteli-mesènquima", procés morfogènic essencial en el desenvolupament embrionari i en els fenòmens metastàsics tumorals.En els mamífers l'activitat d'Snail és modulada per dos mecanismes. (i) En el promotor humà es troben regions definides de resposta a factors repressors, predominants en les cèl·lules epitelials, i elements diferenciats de resposta a inductors de la "transició epiteli-mesènquima". (ii) L'activitat d'Snail és condicionada també per la seva localització subcel·lular, modulada per mecanismes no transcripcionals: la fosforilació d'Snail determina si és o no exclós del nucli. Al citosol no pot actuar com a repressor transcripcional però pot interaccionar amb la xarxa microtubular, que estabilitza i en condiciona el dinamisme. Això coincideix amb l'activació de la GTPasa RhoA i la reorientació dels filaments de vimentina, fets associats a l'adquisició de capacitat migratòria. L'efecte com a repressor transcripcional i la modulació del dinamisme microtubular són possiblement esdeveniments coordinats necessaris per al rol biològic d'Snail en mamífers. / Snail family of transcription factors is fundamental to the "epithelial-mesenchymal transition", morphogenic process essential to embryonic development and metastatic phenomena in tumors.Snail's activity is modulated in two ways in mammals. (i) The human promoter harbors definite regions that respond to repressor factors, which prevail in epithelial cells; and differentiated elements that respond to known inducers of the "epithelial-mesenchymal transition". (ii) Snail's activity is also conditioned by its subcellular localization, mechanism not dependent on its transcriptional control: Snail phosphorylation determines whether Snail is excluded or not from the nucleus. When in the cytosol, Snail is unable to act as a transcriptional repressor, but however binds to the microtubular meshwork, which becomes stabilized and whose dynamism is conditioned as a result. This fact coincides with the activation of the RhoA GTPase and reorientation of vimentin filaments, both phenomena being related to the acquisition of cell motility. The transcriptional repressor and the microtubule dynamics effects are probably two coordinated events necessary to Snail's biological role in mammals. Polimerització in vitro PKCzeta PKC atípica (Protein Kinase C) P65 Promotor Nocodazol NF-kB/Dorsal (Nuclear Factor Kappa-B) NES (seqüència d'export nuclear) Microtúbuls detirosinats Microtúbuls Metàstasi Invasió Adenocarcinoma ARNm Beta-catenina Centrosoma CLIP-170 Cancer Cresta neural CRM1 (Chromosome Region Maintenance 1) Despolimerització microtúbuls Dit de Zenc atípic Dit de Zenc Domini ric en Serines Ebox E-cadherina Espais intercromatínics Estabilització microtúbuls Export nuclear Filaments intermediaris Fosforilació Gastrulació GFP (Green Fluorescent Protein) Heterocarions ILK (Integrin Linked Kinase) Promotor Reconstrucció fronts invasius Regulació Retrogen RhoA GTPasa SC35 Slug SNAG (domini) SNAI1L Snail Speckles nuclears Time-lapse Èster de forbol Transició Epiteli-Mesènquima Transcripció / Transcripcional U2AF65 Vimentina 57

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