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PubMed2000

A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a "shortcut" mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb/mediator proteins of the holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb/mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals.

Bacterial Proteins↗
PubMed1991

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage.

Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD') that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at approximately 180 copies per uninduced cell and was measured at approximately 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with approximately 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.

Bacterial Proteins↗
PubMed1985

Frameshift mutagenesis by 9-aminoacridine and ICR191 in Escherichia coli: effects of uvrB, recA and lexA mutations and of plasmid pKM101.

We have studied the effects of different repair capacities on reversion of two Escherichia coli strains (lacZ19124 and lacZ19136) by 9-aminoacridine (9AA) and the acridine half-mustard ICR191. Introduction of a uvrB mutation into these strains led to enhanced ICR191-induced reversion of lacZ19136 and reduced ICR191-induced reversion of lacZ19124. 9AA-induced reversion of lacZ19124 was essentially unchanged while reversion of lacZ19136 was reduced. Plasmid pKM101 reduced reversion of the two markers by each of the mutagens, except in the case of ICR191-induced reversion of the lacZ19124 marker where mutagenesis was slightly enhanced. Mutations in the recA and lexA genes had minimal effects on ICR191- and on 9AA-induced reversion of the lacZ markers; although 9AA-induced reversion of the lacZ19124 marker was somewhat reduced, most of the other results indicated that mutation yields were if anything higher in the recA or lexA backgrounds. Mutagenesis by 9AA and ICR191 would therefore appear to occur independently of the inducible error-prone repair process commonly referred to as SOS repair.

Aminacrine↗
PubMed1996

Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase. Pinpointing the sites of interaction between p47phoz, p67phox and p40phox using the two-hybrid system.

Activation of the superoxide-generating NADPH-oxidase in phagocytic cells requires the assembly of a membrane-bound flavocytochrome b and cytosolic factors p47phox and p67phox under the control of the GTP-binding protein, Rac. A novel cytosolic component p40phox was recently identified. Most of the components of the complex contain SH3 domains and/or polyproline motifs which are likely to mediate protein-protein interactions occurring in the formation of the active NADPH-complex. The two-hybrid system was used to explore associations between the cytosolic factors. Various constructs of p47phox, p67phox and p40phox cDNAs coding for functional domains were inserted into two-hybrid system vectors, expressing fusion proteins either with the DNA binding protein Lex A or with the activation domain of Gal 4. The site of interaction of p67phox with p47phox was restricted to the C-terminal SH3 domain of p67phox and to the polyproline motif of p47phox. The polyproline motif of p47phox was also found to mediate interaction with the SH3 domain of p40phox, as well as intramolecular interaction within p47phox. The site of interation of p67phox with p40phox was found to be in the 150 amino acid stretch between the two SH3 domains of p67phox. As the C-terminal tail of p40phox which interacts with p67phox contains neither a SH3 domain nor a polyproline consensus site, it is concluded that a novel type of interaction occurs between p40phox and p67phox. Taken together, the results of the two-hybrid experiments led us to formulate a model for oxidase activation, induced by phosphorylation, in which p40phox tends to prevent spontaneous activation.

Amino Acid Sequence↗
PubMed2004

Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro.

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.

Bacterial Proteins↗
PubMed1996

The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C.

The atypical PKCs are involved in a number of important cellular functions, including cell proliferation. We report here that the product of the par-4 gene specifically interacts with the regulatory domains of zeta PKC and lambda/LPKC, which dramatically inhibits their enzymatic activity. This is particularly challenging, because expression of par-4 has been shown to correlate with growth inhibition and apoptosis. Results are shown here demonstrating that the expression of par-4 in NIH-3T3 cells induces morphological changes typical of apoptosis, which are abrogated by cotransfection of either wild-type zeta PKC or lambda/LPKC, but not by their respective kinase-inactive mutants. These findings support a role for the atypical PKC subspecies in the control of cell growth and survival.

3T3 Cells↗
PubMed1996

C-terminal domains of general regulatory factors Abf1p and Rap1p in Saccharomyces cerevisiae display functional similarity.

Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription activation of yeast ribosomal protein genes. This functional link prompted us to investigate whether these factors may be functionally interchangeable. We focused on the indispensable C-terminal portions of both factors and performed mutual domain swaps. The functional capacity of the resulting hybrid proteins was subsequently examined using yeast strains conditionally expressing either the ABF1 or the RAP1 gene. Both the Abf1p-Rap1p and the Rap1p-Abf1p fusion proteins were found to be able to complement the growth defect of the respective strains. Furthermore, Abf1p and Rap1p are both able to promote transcription of a reporter gene through a combination of the respective binding site and a T-rich promoter element. These data strongly suggest that the C-terminal domains of Abf1p and Rap1p have, at least partially, identical functions. Finally, a deletion analysis of the so far largely uncharacterized C-terminal domain of Abf1p was performed, which revealed that two regions of 50 amino acids can perform all essential Abf1p functions.

Amino Acid Sequence↗
PubMed1998

FtsK is an essential cell division protein that is localized to the septum and induced as part of the SOS response.

The role of ftsK in the growth of Escherichia coli was examined by turning off its expression. This resulted in smooth filaments without constrictions, indicating that FtsK was required at an early step in septation. Consistent with this, FtsK was found to localize to the septum in 70% of the cells, indicating that it was recruited relatively early in this process. FtsK localization required the function of FtsZ and FtsA but not FtsI and FtsQ. Consistent with this, Z rings were present in FtsK-depleted filaments. Subcellular localization of FtsK confirmed that it was a membrane protein. Only the first 202 amino acids of FtsK were essential for its role in membrane localization, cell division and viability. The expression of ftsK increased as part of the SOS response, and increased expression of ftsK conferred increased resistance to DNA damage.

Bacterial Proteins↗
PubMed2002

The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB.

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C-terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by cross-linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity.

Bacterial Outer Membrane Proteins↗
PubMed2001

The Bacillus subtilis competence transcription factor, ComK, overrides LexA-imposed transcriptional inhibition without physically displacing LexA.

During the development of competence in Bacillus subtilis the recA gene is activated by the competence transcription factor, ComK, which is presumably required to alleviate the transcriptional repression of recA by LexA. To investigate the mechanism by which ComK activates recA transcription we examined the binding of ComK and LexA to the recA promoter in vitro. Using hydroxyl radical protection analyses to establish the location of ComK dimer-binding sites within the recA promoter, we identified four AT-boxes in a configuration unique for ComK-regulated promoters. Gel mobility shift experiments showed that all four ComK dimer-binding sites were occupied at ComK concentrations in the physiological range. In addition, occupation of all ComK-binding sites did not prevent LexA from binding to the recA promoter, despite the fact that the ComK and LexA recognition motifs partially overlap. Although ComK did not replace LexA from the recA promoter, in vitro transcription analyses indicated that the presence of ComK is sufficient to alleviate LexA repression of recA.

Bacillus subtilis↗
PubMed2002

Different transcription factor binding arrays modulate the cAMP responsivity of the phosphoenolpyruvate carboxykinase gene promoter.

The cAMP responsiveness of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is mediated by a cAMP response unit, which includes three CCAAT/enhancer-binding protein (C/EBPs) sites, and a cAMP response element (CRE). Because both the CRE-binding protein and several C/EBP isoforms can to bind to the CRE with similar affinity, a variety of transcription factor bindings arrays in the cAMP response unit are possible that may affect the protein kinase A (PKA) responsivity of the promoter. To explore this issue, we have designed PEPCK promoter variants that have the native cis-elements within the cAMP response unit replaced with one or more LexA- and/or GAL4-binding sites. We also engineered the corresponding C/EBP and CRE-binding protein chimeras, which have their basic region leucine zipper domains replaced with LexA or GAL4 DNA-binding domains. Using this approach, we have reconstituted the PKA responsiveness of permissive PEPCK promoters in hepatoma cells and have characterized the PKA responsivity of the promoter under defined transcription factor occupancy patterns. Furthermore, analysis of deletion mutants of C/EBPalpha indicated that the domains that mediate its constitutive and PKA-inducible activities vary depending on which cis-element it occupies on the PEPCK promoter. These results suggest that promoter context may influence which domains within a transcription factor are employed to mediate transactivation.

Animals↗
PubMed2002

Rhodobacter sphaeroides LexA has dual activity: optimising and repressing recA gene transcription.

Transcription of the Rhodobacter sphaeroides recA promoter (P(recA)) is induced upon DNA damage in a lexA-dependent manner. In vivo experiments demonstrate that LexA protein represses and might also activate transcription of P(recA). Purified R.sphaeroides LexA protein specifically binds the SOS boxes located within the P(recA) region. In vitro transcription analysis, using Escherichia coli RNA polymerase (RNAP), indicated that the presence of LexA may stimulate and repress transcription of P(recA). EMSA and DNase I footprinting experiments show that LexA and RNAP can bind simultaneously to P(recA). At low LexA concentrations it enhances RNAP binding to P(recA), stimulates open complex formation and strand separation beyond the transcription start site. At high LexA concentrations, however, RNAP-promoted strand separation is not observed beyond the +5 region. LexA might repress transcription by interfering with the clearance process instead of blocking the access of RNAP to the promoter region. Based on these findings we propose that the R.sphaeroides LexA protein performs fine tuning of the SOS response, which might provide a physiological advantage by enhancing transcription of SOS genes and delaying full activation of the response.

Bacterial Proteins↗
PubMed1994

CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context.

The yeast CCR4 protein is required for the expression of a number of genes involved in nonfermentative growth, including glucose-repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance. We show here that although CCR4 did not bind DNA under the conditions tested, it was able to activate transcription when fused to a heterologous DNA-binding domain. The transcriptional activation ability of CCR4, in contrast to that of many other activators, was glucose regulated. Two activation domains one of which was glucose responsive and encompassed a glutamine-proline-rich region similar to that found in other eukaryotic transcriptional factors were identified. The two transactivation regions, when separated from the leucine-rich repeat and the C terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C terminus make contacts that link the activation regions to the proper gene context. Native immunoprecipitation of CCR4 revealed that CCR4 was complexed with at least four other proteins. The leucine-rich repeat of CCR4 was both necessary and sufficient for interaction with at least two of these factors. We propose that the leucine-rich repeat links CCR4 through its associated factors to its promoter context at ADH2 and other loci where it is required for proper transcriptional regulation.

Alcohol Dehydrogenase↗
PubMed1995

Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage- and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.

Amino Acid Sequence↗
PubMed1998

ADR1-mediated transcriptional activation requires the presence of an intact TFIID complex.

The yeast transcriptional activator ADR1, which is required for ADH2 and other genes' expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

Alcohol Dehydrogenase↗
PubMed2000

Cbln3, a novel member of the precerebellin family that binds specifically to Cbln1.

Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide cerebellin. Although cerebellin has properties of a conventional neuropeptide, its function is controversial because Cbln1 has structural features characteristic of circulating atypical collagens. Cbln1 is related to the three subunits of the complement C1q complex. Therefore, we hypothesized that Cbln1 participated in analogous heteromeric complexes with precerebellin-related proteins. Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a cDNA encoding a novel Cbln1-related protein, designated Cbln3. The gene encoding cbln3 had the same intron-exon structure as cbln1 but mapped to a different mouse chromosome (14). The deduced amino acid sequence of Cbln3 was 55% identical to Cbln1 and also contained a C1q signature domain and signal sequence for secretion. In addition to binding avidly to Cbln3, Cbln1 also formed homomeric complexes. In contrast, Cbln3 homomeric association was weak. These interactions exhibited specificity because C1qB bound to neither Cbln1 nor Cbln3. Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear nucleus in which it was detected in granule neurons. Because Cbln1 and Cbln3 are coexpressed in the brain and interact avidly, they may function as a secreted heteromeric complex in vivo.

Aging↗
PubMed1991

[Through homologous recombination pathway recA gene takes part in chromosome replication of E. coli initiated by integrated plasmid].

We have reported in our previous paper that replication of E. coli chromosome initiated by the integrated F' plasmid depends on the recA gene. Here we report on work dealing with the role played by recA, recB, recC, lexA alleles in chromosome replication. Our results show that the recA gene takes part in chromosome replication through homologous recombination rather than SOS pathway and that Chi hot spot is not concerned with. They also show that the ATP-dependent dsDNA exonuclease activity of the RecBC enzyme has nothing to do with the recA gene in chromosome replication.

Bacterial Proteins↗