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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using phenyl-azide-mediated photocrosslinking, we show that the alpha carbon of amino acid 2 of the helix-turn-helix motif of bacteriophage lambda Cro is within 12 A of the bottom-strand nucleotides at positions 2 and 3 of the DNA half site in the Cro-DNA complex in solution. This result is in excellent agreement with the crystallographic structure of the Cro-DNA complex. The results of phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction, together with the previously reported results of phenyl-azide-mediated photocrosslinking analysis of CAP-DNA interaction, establish that phenyl-azide-mediated photocrosslinking is generalizable and provide information regarding the structural requirements for phenyl-azide-mediated photocrosslinking. Comparison of the results of phenyl-azide-mediated photocrosslinking to the results of EDTA: iron-mediated affinity cleaving indicates that phenyl-azide-mediated photocrosslinking yields superior resolution.
J Mol Biol 1993 Mar 20
PMID:Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction. 846 59

We have isolated a rat cDNA whose expression suppresses the physiological consequences of the chromosomal disruption of CAP, the gene encoding the adenylyl cyclase-associated protein of Saccharomyces cerevisiae. Yeast CAP is a bifunctional protein: the NH2 terminus is necessary and sufficient for cellular responsiveness to activated RAS proteins, while the COOH terminus is required for normal cellular morphology and growth control. The rat MCH1 cDNA encodes a protein of 474 amino acids that is 36% identical to S. cerevisiae CAP and is capable of suppressing the loss of the COOH-terminal functions of CAP when expressed in yeast. The MCH1 protein therefore appears to be a structural and functional homolog of the yeast cyclase-associated proteins. Northern analysis of MCH1 gene expression shows it to be constitutively expressed in all cell and tissue types examined. The cloning of a rat homolog of CAP, in addition to the cloning of a human CAP homolog by Matviw et al. (Matviw, H., Yu, G., and Young, D. (1992) Mol. Cell. Biol. 12, 5033-5040), demonstrates that both cyclase-associated proteins and their functions may have evolved with mammalian cells.
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PMID:Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae. 851 80

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.
Mol Cell Biol 1996 Feb
PMID:A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization. 855 82

The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop. We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp. Repression of a CAP-independent lac UV5 promoter by O1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O(id)). Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo. Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression. Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp. Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively. Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor.
J Mol Biol 1996 Mar 22
PMID:Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator. 863 56

Treatment of mouse LMTK- cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 micrograms/ml for 7 days prevented cell growth while maintaining viability, with less than 10(-6) cells recovering to form colonies. Pre-treatment of LMTK- cells with R-6G was followed by fusion with enucleated mouse 501-1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAPR). Cybrids and any surviving unfused LMTK- cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAPR marker. Approximately 1 colony per 2 x 10(5) LMTK- cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAPR mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for rho 0 cell lines.
Somat Cell Mol Genet 1996 Jan
PMID:Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells. 864 97

This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coli genome, compared to its distribution in phages and plasmids. At first sight the distribution of GATC words looks random. But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromasome), is used as a theoretical reference, strong biasesare observed. GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp. The last class is the only one present in E. coli parasites. It can be ascribed to the triggering sequences of the long-patch mismatch repair system. The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection. The other classes, which could be targets for a nucleic acid-binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs. Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions. In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions. In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.
J Mol Biol 1996 Apr 05
PMID:Uneven distribution of GATC motifs in the Escherichia coli chromosome, its plasmids and its phages. 864 25

The crystallographic structure of the CAP-DNA complex at 3.0 A resolution has been reported previously. For technical reasons, the reported structure had been determined using a gapped DNA molecule lacking two phosphates important for CAP-DNA interaction. In this work, we report the crystallographic structure of the CAP-DNA complex at 2.5 A resolution using a DNA molecule having all phosphates important for CAP-DNA interaction. The present resolution permits unambiguous identification of amino acid-base and amino acid-phosphate hydrogen bonded contacts in the CAP-DNA complex. In addition, the present resolution permits accurate definition of the kinked DNA conformation in the CAP-DNA complex.
J Mol Biol 1996 Jul 19
PMID:Structure of the CAP-DNA complex at 2.5 angstroms resolution: a complete picture of the protein-DNA interface. 875 2

PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.
Mol Microbiol 1996 May
PMID:A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. 879 75

Transcription factor-induced DNA distortion has become a common theme in eukaryotic gene regulation. A number of techniques have been applied to the study of transcription factor-induced DNA bending and flexibility including electron microscopy, circular permutation gel analysis, helical phasing gel analysis and cyclisation kinetics in solution. We have applied these techniques in order to assess the role that specific DNA sequences and protein domains of transcription factor IIIA (TFIIIA) play in the TFIIIA-induced distortion of the Xenopus 5 S ribosomal RNA gene promoter. Electron spectroscopic imaging analysis of TFIIIA:DNA complexes indicate that TFIIIA binding involves compaction of the 5 S promoter into a precise three-dimensional hairpin-shaped structure. This compaction can be detected utilising circular permutation gel analysis and the distortion results in an apparent bend angle of 55 to 60 degrees near the centre of the TFIIIA binding site. Helical phasing analysis demonstrates that the 60 degrees bend angle as measured by circular permutation can be detected as a static bend directed towards the minor groove between bases +63 and +64 of the 5 S rRNA gene. The amplitude of the TFIIIA:5 S gene phasing signal is similar to the phasing signal obtained utilising bacterial CAP:DNA complexes with bend angles of approximately 90 degrees. These results are supported by phased ligase-mediated cyclisation kinetics in solution. Analysis of DNA deletion constructs indicate that the 5' A block of the internal 5 S gene promoter, which is required for transcriptional activity, is also required for TFIIIA-induced distortion of the 5 S gene promoter. Analysis of the N-terminal papain fragment of TFIIIA indicates that the 34 kDa zinc finger DNA binding domain is sufficient for compaction of the 5 S gene promoter. These results are discussed in relation to the modular model of TFIIIA:DNA interaction in which individual zinc fingers contribute to the protein-induced distortion of the DNA helix and overall DNA binding affinity in a complex, non-additive fashion.
J Mol Biol 1996 Oct 11
PMID:Protein and DNA requirements for the transcription factor IIIA-induced distortion of the 5 S rRNA gene promoter. 887 41

Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins. Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia. This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction. Decreases in transcript levels following inoculation with wild-type Rhizobium sp. NGR234 were more pronounced than with NGR delta nodABC, a mutant deficient in Nod-factor production. Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGR delta nodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent. Application of purified NodNGR factors only affected transcript levels of ext3. The genomic locus of the gene homologous to ext26 (Ext26G) was cloned. In the 5' flanking region, several potential TATA boxes and CAP signals were identified. Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region. Nonetheless, the function of these homologous regions in gene regulation is unknown.
Mol Plant Microbe Interact 1997 Jan
PMID:Rhizobia modulate root-hair-specific expression of extensin genes. 900 73


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