Tamas Gaal - Academia.edu (original) (raw)
Papers by Tamas Gaal
L'invention concerne de nouveaux vecteurs d'expression, et plus particulierement des vect... more L'invention concerne de nouveaux vecteurs d'expression, et plus particulierement des vecteurs d'expression portant un nouveau type de promoteurs permettant l'expression du gene etranger construit a partir du promoteur P2 de l'operon d'ARN ribosomique de E. coli, et de sequences regulatoires de l'operon lac de E. coli. L'invention concerne en outre un procede de construction des vecteurs precites. Le procede est caracterise par la deletion de la plupart des genes de structure de l'operon d'ARN ribosomique de E. coli (operon rrn) construit dans un vecteur adequat, la reunion de la partie d'operon rrn comprenant le promoteur P2 avec la partie initiale de l'operon lac hors des regions -35 et -10, en aval de celles-ci, de maniere a creer un nouveau site de clivage NsiI au point de jonction des sequences provenant des operons rrm et lac, la deletion de l'element de la sequence TGCA interne du site de clivage NsiI nouvellement cree, et la...
Genes & Development, 2016
The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chr... more The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three ...
Journal of Bacteriology, 1996
The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have... more The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.
Journal of Bacteriology, 1993
The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the... more The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimula...
Journal of Bacteriology, 1989
We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1... more We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutio...
Journal of Bacteriology, 1989
Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p accordi... more Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA pro...
Lecture Notes in Computer Science, 2002
Sequentiality is a desirable property of finite state transducers: such transducers are optimal f... more Sequentiality is a desirable property of finite state transducers: such transducers are optimal for time efficiency. Not all transducers are sequentiable. Sequentialization algorithms of finite state transducers do not recognize whether a transducer is sequentiable or not and simply do not ever halt when it is not. Choffrut proved that sequentiality of finite state transducers is decidable. Béal et al. have proposed squaring to decide sequentiality. We propose a different procedure, which, with ε-closure extension, is able to handle letter transducers with arbitrary ε-ambiguities, too. Our algorithm is more economical than squaring, in terms of size. In different cases of non-sequentiability necessary and sufficient conditions of the ambiguity class of the transducer can be observed. These ambiguities can be mapped bijectively to particular basic patterns in the structure of the transducer. These patterns can be recognized, using finite state methods, in any transducer.
Mechanisms of Transcription, 1997
Proceedings of the National Academy of Sciences
Journal of Bacteriology
ABSTRACT
Lecture Notes in Computer Science, 2001
Sequential transducers, introduced by Schützenberger [5], have advantageous computational propert... more Sequential transducers, introduced by Schützenberger [5], have advantageous computational properties. A sequential transducer is deterministic with respect to its input. Not all transducers can be sequentialized: but if one can be, it means time, and, often, space optimality. This article extends the subsequentialization algorithm of Mohri [3,4] for previously untreated classes of transducers. We — change the representation of final
Lecture Notes in Computer Science, 2003
This article presents a new tool, WFSC, for creating, manipulating, and applying weighted finite ... more This article presents a new tool, WFSC, for creating, manipulating, and applying weighted finite state automata. It inherits some powerful features from Xerox's non-weighted XFST tool and represents a continuation of Xerox's work in the field of finite state automata over two decades. The design is generic: algorithms work on abstract components of automata and on a generic abstract semiring, and are independent of their concrete realizations. Applications can access WFSC's functions through an API or create automata through an end-user interface, either from an enumeration of their states and transitions or from rational expressions.
Lecture Notes in Computer Science, 2006
We propose a new model of finite state machine: multi-tape automata with symbol classes and ident... more We propose a new model of finite state machine: multi-tape automata with symbol classes and identity and non-identity constraints (in short M ASCIN). This model generalizes both classical single or multi-tape machines, and machines with extended alphabet. We define this model in terms of a constraint satisfaction problem and discuss a problem occurring when projection is used on the model. Finally, we describe its implementation and results of a performance test.
Protein Expression and Purification, 2010
We report an improved procedure for purification of the omega subunit of Escherichia coli RNA pol... more We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only 2 chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and cooverexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.
L'invention concerne de nouveaux vecteurs d'expression, et plus particulierement des vect... more L'invention concerne de nouveaux vecteurs d'expression, et plus particulierement des vecteurs d'expression portant un nouveau type de promoteurs permettant l'expression du gene etranger construit a partir du promoteur P2 de l'operon d'ARN ribosomique de E. coli, et de sequences regulatoires de l'operon lac de E. coli. L'invention concerne en outre un procede de construction des vecteurs precites. Le procede est caracterise par la deletion de la plupart des genes de structure de l'operon d'ARN ribosomique de E. coli (operon rrn) construit dans un vecteur adequat, la reunion de la partie d'operon rrn comprenant le promoteur P2 avec la partie initiale de l'operon lac hors des regions -35 et -10, en aval de celles-ci, de maniere a creer un nouveau site de clivage NsiI au point de jonction des sequences provenant des operons rrm et lac, la deletion de l'element de la sequence TGCA interne du site de clivage NsiI nouvellement cree, et la...
Genes & Development, 2016
The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chr... more The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three ...
Journal of Bacteriology, 1996
The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have... more The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.
Journal of Bacteriology, 1993
The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the... more The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimula...
Journal of Bacteriology, 1989
We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1... more We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutio...
Journal of Bacteriology, 1989
Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p accordi... more Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA pro...
Lecture Notes in Computer Science, 2002
Sequentiality is a desirable property of finite state transducers: such transducers are optimal f... more Sequentiality is a desirable property of finite state transducers: such transducers are optimal for time efficiency. Not all transducers are sequentiable. Sequentialization algorithms of finite state transducers do not recognize whether a transducer is sequentiable or not and simply do not ever halt when it is not. Choffrut proved that sequentiality of finite state transducers is decidable. Béal et al. have proposed squaring to decide sequentiality. We propose a different procedure, which, with ε-closure extension, is able to handle letter transducers with arbitrary ε-ambiguities, too. Our algorithm is more economical than squaring, in terms of size. In different cases of non-sequentiability necessary and sufficient conditions of the ambiguity class of the transducer can be observed. These ambiguities can be mapped bijectively to particular basic patterns in the structure of the transducer. These patterns can be recognized, using finite state methods, in any transducer.
Mechanisms of Transcription, 1997
Proceedings of the National Academy of Sciences
Journal of Bacteriology
ABSTRACT
Lecture Notes in Computer Science, 2001
Sequential transducers, introduced by Schützenberger [5], have advantageous computational propert... more Sequential transducers, introduced by Schützenberger [5], have advantageous computational properties. A sequential transducer is deterministic with respect to its input. Not all transducers can be sequentialized: but if one can be, it means time, and, often, space optimality. This article extends the subsequentialization algorithm of Mohri [3,4] for previously untreated classes of transducers. We — change the representation of final
Lecture Notes in Computer Science, 2003
This article presents a new tool, WFSC, for creating, manipulating, and applying weighted finite ... more This article presents a new tool, WFSC, for creating, manipulating, and applying weighted finite state automata. It inherits some powerful features from Xerox's non-weighted XFST tool and represents a continuation of Xerox's work in the field of finite state automata over two decades. The design is generic: algorithms work on abstract components of automata and on a generic abstract semiring, and are independent of their concrete realizations. Applications can access WFSC's functions through an API or create automata through an end-user interface, either from an enumeration of their states and transitions or from rational expressions.
Lecture Notes in Computer Science, 2006
We propose a new model of finite state machine: multi-tape automata with symbol classes and ident... more We propose a new model of finite state machine: multi-tape automata with symbol classes and identity and non-identity constraints (in short M ASCIN). This model generalizes both classical single or multi-tape machines, and machines with extended alphabet. We define this model in terms of a constraint satisfaction problem and discuss a problem occurring when projection is used on the model. Finally, we describe its implementation and results of a performance test.
Protein Expression and Purification, 2010
We report an improved procedure for purification of the omega subunit of Escherichia coli RNA pol... more We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only 2 chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and cooverexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.