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PubMed1991

Generating yeast transcriptional activators containing no yeast protein sequences.

We previously reported that roughly 1% of the short peptides encoded by Escherichia coli genomic DNA fragments act as transcriptional activating regions in yeast when fused to GAL4(1-147), a DNA-binding portion of the yeast transcriptional activator GAL4 (ref. 1). Struhl questioned the conclusion that we had identified new transcriptional activating sequences that function in the absence of yeast transcriptional activating sequences. His criticism was based on two considerations: first, GAL4(1-147) contains an acidic segment (and subsequent experiments have shown that this region contains a weak activating region in vitro); second, attempts to isolate new activating regions failed when the DNA-binding domain of a bacterial repressor, LexA(1-87), was used as the DNA-binding unit. We report here a repeat of our original experiment using the complete LexA molecule LexA(1-202) as the DNA-binding region, instead of GAL4(1-147) or LexA(1-87). We find that, as in the original experiment, about 1% of the short peptides encoded by E. coli genomic fragments act as transcriptional activating regions when fused to intact LexA. All of the new activating regions whose sequences we determined bore an excess of acidic amino acids (see Table 1).

Amino Acid Sequence↗
PubMed1995

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.

Bacterial Proteins↗
PubMed2005

Genomic analysis of LexA binding reveals the permissive nature of the Escherichia coli genome and identifies unconventional target sites.

Genomes of eukaryotic organisms are packaged into nucleosomes that restrict the binding of transcription factors to accessible regions. Bacteria do not contain histones, but they have nucleoid-associated proteins that have been proposed to function analogously. Here, we combine chromatin immunoprecipitation and high-density oligonucleotide microarrays to define the in vivo DNA targets of the LexA transcriptional repressor in Escherichia coli. We demonstrate a near-universal relationship between the presence of a LexA sequence motif, LexA binding in vitro, and LexA binding in vivo, suggesting that a suitable recognition site for LexA is sufficient for binding in vivo. Consistent with this observation, LexA binds comparably to ectopic target sites introduced at various positions in the genome. We also identify approximately 20 novel LexA targets that lack a canonical LexA sequence motif, are not bound by LexA in vitro, and presumably require an additional factor for binding in vivo. Our results indicate that, unlike eukaryotic genomes, the E. coli genome is permissive to transcription factor binding. The permissive nature of the E. coli genome has important consequences for the nature of transcriptional regulatory proteins, biological specificity, and evolution.

Bacterial Proteins↗
PubMed1993

Genetic evidence for transcriptional activation by the yeast IME1 gene product.

IME1 is required in yeast for meiosis and for expression of IME2 and other early meiotic genes. IME1 is a 360-amino acid polypeptide with central and C-terminal tyrosine-rich regions. We report here that a fusion protein composed of the lexA DNA-binding domain and IME1 activates transcription in vivo of a reporter gene containing upstream lexA binding sites. Activation by the fusion protein shares several features with natural IME1 activity: both are dependent on the RIM11 gene product; both are impaired by the same ime1 missense mutations; both are restored by intragenic suppressors. The central tyrosine-rich region is sufficient to activate transcription when fused to lexA. Deletion of this putative activation domain results in a defective IME1 derivative. Function of the deletion derivative is restored by fusion to the acidic Herpesvirus VP16 activation domain. The C-terminal tyrosine-rich region is dispensable for transcriptional activation; rather it renders activation dependent upon starvation and RIM11. Immunofluorescence studies indicate that an IME1-lacZ fusion protein is concentrated in the nucleus. These observations are consistent with a model in which IME1 normally stimulates IME2 expression by providing a transcriptional activation domain at the IME2 5' regulatory region.

Bacterial Proteins↗
PubMed1989

Yeast upstream activator protein GCN4 can stimulate transcription when its binding site replaces the TATA element.

We replaced the required TATA element of a yeast gal-his3 promoter by a binding site for GCN4, a protein that normally activates transcription when bound upstream of a TATA element. Surprisingly, GCN4 efficiently activates his3 transcription from wild-type initiation sites, though in a pattern associated with constitutive his3 transcription rather than GCN4 upstream activation through a TATA element. Transcriptional stimulation by GCN4 requires both the DNA-binding domain and the acidic activation function but is not affected by changing the spacing or helical relationship between the GCN4 binding site and the mRNA start sites. GCN4 is not sufficient for this TATA-independent activation; a sequence in the gal fragment distinct from the GAL4 binding sites is also required. Thus, GCN4 functions both when bound upstream of a TATA element and also when bound at the position of a TATA element. In the latter case, we suggest the possibility that GCN4 might be able to stimulate transcription by an alternate mechanism that does not involve a conventional TATA-binding transcription factor.

Bacterial Proteins↗
PubMed2000

Macromolecular assemblage of aminoacyl-tRNA synthetases: quantitative analysis of protein-protein interactions and mechanism of complex assembly.

The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches. This macromolecular assemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the seven monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 proteins. The scaffold p38 protein was expressed in Escherichia coli and purified to homogeneity as a His-tagged protein. The different components of the complex were shown to associate in vitro with p38 immobilized on Ni(2+)-coated plates. Interactions between peripheral enzymes and p38 are referred to as central interactions, as opposed to lateral interactions between peripheral enzymes. Kinetic parameters of the interactions were determined by the means of a biosensor-based approach. The two dimeric proteins LysRS and AspRS were found to tightly bind to p38, with a K(d) value of 0.3 and 4.7 nM, respectively. These interactions involved the catalytic core of the enzymes. By contrast, binding of ArgRS or GlnRS to p38 was much weaker (>5 microM). ArgRS and p43, two peripheral components, were shown to interact with moderate affinity (K(d)=93 nM). Since all the components of the complex are tightly associated within this particle, lateral interactions were believed to contribute to the stabilization of this assemblage. Using an in vitro binding assay, concomitant association of several components of the complex on immobilized p38 could be demonstrated, and revealed the involvement of synergistic effects for association of weakly interacting proteins. Taking into account the possible synergy between central and lateral contributions, a sub-complex containing p38, p43, ArgRS and GlnRS was reconstituted in vitro. These data provide compelling evidence for an ordered and concerted mechanism of complex assembly.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Mutational analysis of transcriptional activation by the bovine papillomavirus type 1 E6.

While the bovine papillomavirus type 1 (BPV-1) E6 induces tumorigenic transformation of murine C127 cells, it does not bind or promote the degradation of p53. We recently showed the cellular protein ERC-55/E6BP binds BPV-1 E6 as well as the cancer-related human papillomavirus (HPV) E6 proteins. BPV-1 E6 also binds E6-AP, a ubiquitin ligase necessary for HPV E6-induced p53 degradation. We previously reported that the transforming activity of a set of BPV-1 E6 mutants correlated with their E6BP-binding ability. Another function of BPV-1 E6 is stimulation of transcription when targeted to a promoter, although cellular promoters responsive to BPV-1 E6 have not been identified. To examine whether its transcriptional function is required for oncogenic activity, or is related to its interactions with E6-AP or E6BP, a series of BPV-1 E6 mutants were analyzed as fusions to a sequence-specific DNA binding domain for activity in yeast and in mammalian cells. We show that some transformation defective mutants retained substantial levels of transcriptional activation activity. These mutants also distinguish transcriptional activation from E6-AP and E6BP binding. These results suggest the transcriptional activation function of BPV-1 E6 is not sufficient for cell transformation.

3T3 Cells↗
PubMed1985

Involvement of DNA lesions and SOS functions in 5-bromouracil-induced mutagenesis.

Mutagenesis resulting from incorporation of 5-bromouracil (BU) in the DNA of E. coli K12 proceeds largely (approximately 80%) via misrepair of the lesions resulting from incorporation of the analogue. The premutational lesions are due principally to dehalogenation of incorporated BU residues, leading to formation of uracil residues, and removal of these by uracil-DNA glycosylase with formation of apyrimidinic sites. In the xthA mutant, defective in AP endonuclease, there is a several-fold increase in the frequency of BU-induced mutations, underlining the importance of AP sites in BU-induced mutagenesis. Premutational lesions undergo mutation frequency decline (MFD), which is subject to delay in the xthA mutant, pointing to some role of AP endonuclease in MFD, and further supporting involvement of AP sites in BU-induced mutagenesis. Efficient BU mutagenesis is dependent on the functions of the genes recA and umuC and non-mutated lexA protein.

Bacterial Proteins↗
PubMed2001

Okadaic acid induces transcription of junB through a CCAAT box and NF-Y.

The shellfish toxin, okadaic acid (OA), is a potent tumor promoter that induces expression of the proto-oncogene junB in mouse keratinocyte 308 cells. Here we show, through deletion analysis of the junB promoter, that sequences near the TATA box conferred transcriptional induction by OA. Transient transfections of luciferase constructs bearing the junB promoter with single mutations in various cis elements demonstrated that a promoter containing a mutated CCAAT box could not be induced by OA. When this CCAAT box was inserted into a heterologous promoter construct, OA induction was dependent on an intact CCAAT box. Flanking cis elements located near the CCAAT box, although not required for OA inducibility, did play a role in the basal level of transcription. NF-Y was shown by EMSA to bind to the CCAAT box. OA induction from the junB CCAAT box was blocked by dominant negative NF-YA as well as the CCAAT box-dependent anticancer drug, ET-473. Expression of a lexA/NF-YA chimeric protein demonstrated that OA induction was dependent on the binding of NF-Y family members. These studies demonstrate that OA can mediate transcriptional activation of junB through the classical CCAAT box and that transcription factor NF-Y plays a functional role in the induction.

Animals↗
PubMed2004

Bacillus subtilis transcriptional regulators interaction.

Bacillus subtilis aprE gene codes for the extracellular protease subtilisin. Its expression is controlled by AbrB, DegU, Hpr, SinI, SinR and Spo0A transition state protein regulators. To determine in vivo the protein-protein interactions among these regulators, we used the LexA-based bacterial genetic two-hybrid system. Our results show homo-dimerization to all the analyzed proteins and hetero-dimerization between SinR-SinI and SinR-Hpr.

Bacillus subtilis↗
PubMed2001

Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli.

Type I DNA topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism. Escherichia coli possesses two type I topoisomerase activities, DNA topoisomerase I (Topo I) and III (Topo III). The gene encoding Topo III (topB) can be deleted without affecting cell viability. Cells possessing a deletion of the gene encoding Topo I (topA) are only viable in the presence of an additional compensatory mutation. In the presence of compensatory mutations, Topo I deletion strains grow normally; however, if Topo III activity is repressed in these cells, they filament extensively and possess an abnormal nucleoid structure. These defects can be suppressed by the deletion of the recA gene, suggesting that these enzymes may be involved in RecA-mediated recombination and may specifically resolve recombination intermediates before partitioning.

Anaphase↗
PubMed1998

Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae.

The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression. Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells. In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes. Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex. Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway. The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5. These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.

Bacterial Proteins↗
PubMed1996

Switching transcription on and off during the yeast cell cycle: Cln/Cdc28 kinases activate bound transcription factor SBF (Swi4/Swi6) at start, whereas Clb/Cdc28 kinases displace it from the promoter in G2.

When yeast cells reach a critical size in late G1 they simultaneously start budding, initiate DNA synthesis, and activate transcription of a set of genes that includes G1 cyclins CLN1, CLN2, and many DNA synthesis genes. Cell cycle-regulated expression of CLN1, CLN2 genes is attributable to the heteromeric transcription factor complex SBF. SBF is composed of Swi4 and Swi6 and binds to the promoters of CLN1 and CLN2. Different cyclin-Cdc28 complexes have different effects on late G1-specific transcription. Activation of transcription at the G1/S boundary requires Cdc28 and one of the G1 cyclins Cln1-Cln3, whereas repression of SBF-regulated genes in G2 requires the association of Cdc28 with G2-specific cyclins Clb1-Clb4. Using in vivo genomic footprinting, we show that SBF (Swi4/Swi6) binding to SCB elements (Swi4/Swi6 cell cycle box) in the CLN2 promoter is cell cycle regulated. SBF binds to the promoter prior to the activation of transcription in late G1, suggesting that Cln/Cdc28 kinase regulates the ability of previously bound SBF to activate transcription. In contrast, SBF dissociates from the CLN2 promoter when transcription is repressed during G2 and M phases, suggesting that Clb1-Clb4 repress SBF activity by inhibiting its DNA-binding activity. Switching transcription on and off by different mechanisms could be important to ensure that Clns are activated only once per cell cycle and could be a conserved feature of cell cycle-regulated transcription.

Bacterial Proteins↗
PubMed1995

Insertion of inverted Ter sites into the terminus region of the Escherichia coli chromosome delays completion of DNA replication and disrupts the cell cycle.

To investigate the co-ordination between DNA replication and cell division, we have disrupted the DNA-replication cycle of Escherichia coli by inserting inverted Ter sites into the terminus region to delay completion of the chromosome. The inverted Ter sites (designated InvTer::spcr) were initially inserted into the chromosome of a delta tus strain to allow unrestrained chromosomal replication. We then introduced a functional tus gene by transforming the InvTer::spcr strain with a plasmid carrying the tus gene under control of an arabinose-inducible promoter. In the presence of 0.2% arabinose, the cells formed long filaments, suggesting that activation of the inverted Ter sites by Tus arrested DNA replication and delayed the onset of cell division. Induction of sfiA, a gene in the SOS regulon, was observed following arrest of DNA replication; however, when a sfiB114 allele was introduced into InvTer::spcr strain, long filaments were still formed, suggesting that the sfi-independent pathway also caused filamentation. Either recA::camr or lexA3 alleles suppressed filamentation when introduced in the InvTer strain. Interestingly, in both the recA::camr and lexA3 mutants, virtually all cells had a nucleoid, suggesting that cell division was proceeding even though DNA replication was not complete. These results suggest that DNA replication and cell division are uncoupled when recA is inactivated or when genes repressed by LexA cannot be induced.

Bacterial Proteins↗
PubMed1992

The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression.

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.

Amino Acid Sequence↗
PubMed1996

Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein.

We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein. A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed. The fusion protein, IN1-288/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN). Using an in vitro integration assay that included multiple DNA fragment as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein. No bias was observed when the LexA-binding sequence was absent, when the fusion protein was replaced by WT IN, or when LexA protein was added in the reaction containing IN1-288/LA. A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence. The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase. The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.

Bacterial Proteins↗
PubMed2000

Meiotic segregation, synapsis, and recombination checkpoint functions require physical interaction between the chromosomal proteins Red1p and Hop1p.

In yeast, HOP1 and RED1 are required during meiosis for proper chromosome segregation and the consequent formation of viable spores. Mutations in either HOP1 or RED1 create unique as well as overlapping phenotypes, indicating that the two proteins act alone as well as in concert with each other. To understand which meiotic processes specifically require Red1p-Hop1p hetero-oligomers, a novel genetic screen was used to identify a single-point mutation of RED1, red1-K348E, that separates Hop1p binding from Red1p homo-oligomerization. The Red1-K348E protein is stable, phosphorylated in a manner equivalent to Red1p, and undergoes efficient homo-oligomerization; however, its ability to interact with Hop1p both by two-hybrid and coimmunoprecipitation assays is greatly reduced. Overexpression of HOP1 specifically suppresses red1-K348E, supporting the idea that the only defect in the protein is a reduced affinity for Hop1p. red1-K348E mutants exhibit reduced levels of crossing over and spore viability and fail to undergo chromosome synapsis, thereby implicating a role for Red1p-Hop1p hetero-oligomers in these processes. Furthermore, red1-K348E suppresses the sae2/com1 defects in meiotic progression and sporulation, indicating a previously unknown role for HOP1 in the meiotic recombination checkpoint.

Alleles↗