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Query: UNIPROT:P06889 (Mol)
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The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.
Mol Microbiol 2000 Apr
PMID:Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop. 1076 Jan 79

The Saccharomyces cerevisiae genes PHO80 and PHO85 encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulate PHO5 gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomal pho85 gene disruption in which the HIS3 gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement the pho85::HIS3 gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented the pho85 mutation in yeast strains in which the entire PHO85 coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on the pho85 coding region which remained in the mutant pho85::HIS3 chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.
Mol Gen Genet 2000 Mar
PMID:Intergenic complementation truncation mutants of cyclin-dependent kinase. 1077 40

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma 54 factor (RNAP.sigma(54)) from the closed complex (RNAP.sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer sequence, interaction of this complex with promoter-bound RNAP.sigma(54) via DNA looping, and hydrolysis of ATP. We have used this system to study the influence of the DNA conformation on the transcription activation rate in single-round transcription experiments with superhelical plasmids as well as linearized templates. Most of the templates had an intrinsically curved DNA sequence between the enhancer and the promoter and differed with respect to the location of the curvature and the distance between the two DNA sites. The following results were obtained: (i) a ten- to 60-fold higher activation rate was observed with the superhelical templates as compared to the linearized conformation; (ii) the presence of an intrinsically curved DNA sequence increased the activation rate of linear templates about five times; (iii) no systematic effect for the presence and/or location of the inserted curved sequence was observed for the superhelical templates. However, the transcription activation rate varied up to a factor of 10 between some of the constructs. (iv) Differences in the distance between enhancer and promoter had little effect for the superhelical templates studied. The results were compared with theoretical calculations for the dependence of the contact probability between enhancer and promoter expressed as the molar local concentration j(M). A correlation of j(M) with the transcription activation rate was observed for values of 10(-8) M<j(M)<10(-6) M and a kinetic model for NtrC-P-catalyzed open complex formation was developed.
J Mol Biol 2000 Jul 21
PMID:The effect of the DNA conformation on the rate of NtrC activated transcription of Escherichia coli RNA polymerase.sigma(54) holoenzyme. 1089 Dec 65

Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing the HIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at the CHA1 promoter. Finally, we show that activation of the GAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.
Mol Cell Biol 2000 Aug
PMID:Artificially recruited TATA-binding protein fails to remodel chromatin and does not activate three promoters that require chromatin remodeling. 1091 68

Chromatin remodeling complexes help regulate the structure of chromatin to facilitate transcription. The multisubunit human (h) SWI-SNF complex has been shown to remodel mono- and polynucleosome templates in an ATP-dependent manner. The isolated hSWI-SNF ATPase subunits BRG1 and hBRM also have these activities. The intact complex has been shown to produce a stable remodeled dimer of mononucleosomes as a product. Here we show that the hSWI-SNF ATPases alone can also produce this product. In addition, we show that hSWI-SNF and its ATPases have the ability to transfer histone octamers from donor nucleosomes to acceptor DNA. These two reactions are characterized and compared. Our results are consistent with both products of SWI-SNF action being formed as alternative outcomes of a single remodeling mechanism. The ability of the isolated ATPase subunits to catalyze these reactions suggests that these subunits play a key role in determining the mechanistic capabilities of the SWI-SNF family of remodeling complexes.
Mol Cell Biol 2000 Sep
PMID:Octamer transfer and creation of stably remodeled nucleosomes by human SWI-SNF and its isolated ATPases. 1093 15

Oncoproteins from DNA tumor viruses such as adenovirus E1a, simian virus 40 T antigen, and human papillomavirus E7 contain an LXCXE sequence, which they use to bind the retinoblastoma protein (Rb) and inhibit its function. Cellular proteins such as histone deacetylases 1 and 2 (HDAC1 and -2) also contain an LXCXE-like sequence, which they use to interact with Rb. The LXCXE binding site in Rb was mutated to assess its role in Rb function. These mutations inhibited binding to HDAC1 and -2, which each contain an LXCXE-like sequence, but had no effect on binding to HDAC3, which lacks an LXCXE-like sequence. Mutation of the LXCXE binding site inhibited active transcriptional repression by Rb and prevented it from effectively repressing the cyclin E and A gene promoters. In contrast, mutations in the LXCXE binding site did not prevent Rb from binding and inactivating E2F. Thus, the LXCXE mutations appear to separate Rb's ability to bind and inactivate E2F from its ability to efficiently recruit HDAC1 and -2 and actively repress transcription. In transient assays, several of the LXCXE binding site mutants caused an increase in the percentage of cells in G(1) by flow cytometry, suggesting that they can arrest cells. However, this effect was transient, as none of the mutants affected cell proliferation in longer-term assays examining bromodeoxyuridine incorporation or colony formation. Our results then suggest that the LXCXE binding site is important for full Rb function. Mutation of the LXCXE binding site does not inhibit binding of the BRG1 ATPase component of the SWI/SNF nucleosome remodeling complex, which has been shown previously to be important for Rb function. Indeed, overexpression of BRG1 and Rb in cells deficient for the proteins led to stable growth inhibition, suggesting a cooperative role for SWI/SNF and the LXCXE binding site in efficient Rb function.
Mol Cell Biol 2000 Sep
PMID:Role of the LXCXE binding site in Rb function. 1095 76

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator, ICP4. A reduction of more than 80% in the expression level of ICP4 and a reduction of about 1000-fold in viral growth were observed in cells that stably expressed the ribozyme. In contrast, a reduction of less than 10 % in ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, M1GS ribozyme is highly effective in inhibiting HSV-1 growth and can be used as a general gene-targeting agent for anti-HSV applications.
J Mol Biol 2000 Aug 25
PMID:A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1. 1096 88

Here we have examined HIV-1 nucleosome remodeling upon the binding of transcription factors and the SWI/SNF complex using a novel approach. The approach combines UV laser protein-DNA crosslinking, electrophoretic mobility-shift analysis and DNase I protection analysis with immunochemical techniques. It was found that single activator-bound HIV-1 nucleosomes exhibit very weak perturbation in histone NH(2) tail-DNA interactions. However, the simultaneous binding of the transcription activators Sp1, NF-kB1, LEF-1 and USF synergistically increased the release of histone NH(2) tails from nucleosomal DNA. In contrast, the binding of SWI/SNF complex to HIV-1 nucleosome disrupted structured histone domain-DNA contacts, but not histone NH(2) tail-DNA interactions. Stable remodeled nucleosomes, (obtained after detachment of SWI/SNF), displayed identical structural alterations with those bound to SWI/SNF. These results demonstrate a different in vitro remodeling of the HIV-1 nucleosome upon the binding of multiple transcription activators and of SWI/SNF complex.
J Mol Biol 2000 Sep 15
PMID:Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex binding. 1097 Jul 36

Analysis of the Neisseria gonorrhoeae DNA sequence database revealed the presence of two genes, one encoding a protein predicted to be 37. 5% identical (50% similar) in amino acid sequence to the Escherichia coli FNR protein and the other encoding a protein 41% and 42% identical (54 and 51% sequence similarity) to the E. coli NarL and NarP proteins respectively. Both genes have been cloned into E. coli and insertionally inactivated in vitro. The mutated genes have been transformed into gonococci and recombined into the chromosome. The fnr mutation totally abolished and the narP mutation severely diminished the ability of gonococci to: (i) grow anaerobically; (ii) adapt to oxygen-limited growth; (iii) initiate transcription from the aniA promoter (which directs the expression of a copper-containing nitrite reductase, AniA, in response to the presence of nitrite); and (iv) reduce nitrite during growth in oxygen-limited media. The product of nitrite reduction was identified to be nitrous oxide. Immediately upstream of the narL/narP gene is an open reading frame that, if translated, would encode a homologue of the E. coli nitrate- and nitrite-sensing proteins NarX and NarQ. As transcription from the aniA promoter was not activated during oxygen-limited growth in the presence of nitrate, the gonococcal two-component regulatory system is designated NarQ-NarP rather than NarX-NarL. As far as we are aware, this is the first well-documented example of a two-component regulatory system working in partnership with a transcription activator in pathogenic neisseria. A 45 kDa c-type cytochrome that was synthesized during oxygen-limited, but not during oxygen sufficient, growth was identified as a homologue of cytochrome c peroxidases (CCP) of other bacteria. The gene for this cytochrome, designated ccp, was located, and its regulatory region was cloned into the promoter probe vector pLES94. Transcription from the ccp promoter was repressed during aerobic growth and induced during oxygen-limited growth and was totally FNR dependent, suggesting that the gonococcal FNR protein is a transcription activator of at least two genes. However, unlike AniA, synthesis of the CCP homologue was insensitive to the presence of nitrite during oxygen-limited growth.
Mol Microbiol 2000 Aug
PMID:Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. 1097 6

Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) during transcription activation at FNR-regulated promoters. Using an alphaCTD alanine scan mutant library, we have identified the residues of alphaCTD that are important for FNR-dependent transcription activation. Residues Asp-305, Gly-315, Arg-317, Leu-318 and Asp-319 are proposed to be the key residues in the contact site on alphaCTD for Activating Region 1 of FNR. In previous work, it had been shown that Activating Region 1 of FNR is a large surface-exposed patch and that the two crucial amino acid residues are Thr-118 and Ser-187. In this work, we have constructed Arg-118 FNR and Arg-187 FNR and shown that both FNR derivatives are defective in transcription activation. However, the activity of FNR carrying Arg-118 can be partially restored by substitutions of Lys-304 in alphaCTD. Similarly, the activity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in alphaCTD. The specificity of the restoration suggests that, during transcription activation by FNR, the side-chain of residue 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in alphaCTD. Similarly, the side-chain of residue 187 in Activating Region 1 of FNR is located close to Arg-317 and Leu-318 in alphaCTD. These results can be used to model the interface between Activating Region 1 of FNR and its contact target in alphaCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and alphaCTD.
Mol Microbiol 2000 Sep
PMID:Analysis of interactions between Activating Region 1 of Escherichia coli FNR protein and the C-terminal domain of the RNA polymerase alpha subunit: use of alanine scanning and suppression genetics. 1097 22


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