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

The interaction of the Klebsiella pneumoniae NifA protein, a sigma 54-dependent activator, with the nifE and nifU promoters was analysed. At these promoters NifA established contacts in addition to those predicted by the minimal formulation NifA binding site (5'-TGT-N10-ACA). The positions of the contacts indicate that bound NifA molecules could assemble to form an oligomer. At both promoters contacts with NifA are made predominantly on one face of the DNA helix, and all contacts appear necessary for full activation by NifA. The close contacts made by NifA appear to be made by the DNA-binding domain of NifA. This domain shows specific DNA-binding activity in vitro. The binding of NifA to one site in the nifU promoter depends upon occupancy of additional upstream sequences by NifA. At the nifE promoter NifA binds adjacent to an integration host factor (IHF) binding site, but in contrast to results obtained with the nifU promoter IHF does not diminish nifE promoter occupancy by NifA. The IHF requirement for efficient in vivo activation of the nifU promoter by NifA was greater than that of the nifE promoter. Accordingly, the affinity of IHF for the nifU promoter is higher than for the nifE promoter. Amongst promoters utilizing the sigma 54 holoenzyme, the nifE promoter appears somewhat atypical in having the activator bound at around position -74 rather than the usual 100 base-pairs or more upstream from the transcription start site.
J Mol Biol 1991 Aug 20
PMID:Organization and function of binding sites for the transcriptional activator NifA in the Klebsiella pneumoniae nifE and nifU promoters. 188 Aug 4

The anticancer drug daunomycin has been co-crystallized with the hexanucleotide duplex sequences d(TGTACA) and d(TGATCA) and single crystal X-ray diffraction studies of these two complexes have been carried out. Structure solution of the d(TGTACA) and d(TGATCA) complexes to 1.6 and 1.7 Angstrom resolution, respectively, shows two daunomycin molecules bound to the DNA hexamer. Binding occurs via intercalation of the drug chromophore at the d(TpG) step, and hydrogen bonding interactions involving the drug, DNA and solvent molecules. The daunomycin sugar is located in the minor groove of the DNA hexamer and is stabilized by hydrogen bonds between the amino group of the sugar and functional groups on the floor of the groove. The amino sugar of the d(TGATCA) duplex interacts directly with the DNA sequence, while in the d(TGTACA) duplex, the interaction is via solvent molecules. Two other complexes d(CGTACG)-daunomycin and d(CGATCG)-daunomycin have previously been structurally characterized. Comparison of the four structures with daunomycin bound to the triplet sequences 5'TGT, 5'TGA, 5'CGT and 5'CGA reveals changes in the conformation of both the DNA hexamer and the daunomycin upon complexation, as well as the hydrogen bonding and van der Waals' interactions.
J Mol Biol 1991 Nov 20
PMID:DNA-drug interactions. The crystal structures of d(TGTACA) and d(TGATCA) complexed with daunomycin. 196 Jul 20

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Oct
PMID:The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon). 217 17

Interaction of highly purified glucocorticoid receptor complex (GIRC) with synthetic DNA-fragment of mouse metallotionein 1 gene promoter from -209 to -252 b.p. (MTwt) was investigated. By means of nitrocellulose filter binding assay this fragment was shown to contain specific GIRC-binding site. In order to analyse the fine structure of the site, two variants of this DNA-fragment were synthesized and used in gel retardation assay. GIRC specific binding was shown to retain throughout interaction with the fragment in which all base pairs in the surroundings of generally accepted GIRC-binding site consensus G--ACA---TGTTCT C--TGT---ACAAGA were substituted by means of transitions, but it was weaker than the GIRC-binding with MTwt, where the mentioned consensus was situated in the natural surroundings. Complete loss of the GIRC-binding ability was observed when five CG pairs were substituted by AT ones. Two of the CG pairs belonged to the mentioned consensus. Comparison of the data obtained with results of computer analysis allows to consider the consensus as a "core" of GIRC-binding site, flanked with additional elements, interacting with GIRC.
Mol Biol (Mosk)
PMID:[Identification of the glucocorticoid receptor binding site at the 5'-flanking region of mouse metallothionein I gene: the effect of base substitutions on binding efficiency]. 225 Jun 77

A 9.35 kbp element with long terminal direct repeats (LTRs) of 2.4 kbp has been characterized from the large genome of Lilium henryi. The organization of the element, named del, was examined in 20 fragments from a genomic DNA library constructed using phage EMBL3. Five fragments apparently contained full 9.35 kbp elements while in 11 del was recovered in part. Two clones carried tandem arrangements of del, with single LTRs and internal sequences alternating, and one insert contained a solo LTR. Evidence suggests that these arrangements, which can arise by unequal crossing over, have a genomic rather than a cloning origin. Finally, one cloned fragment had a complex del arrangement yet to be fully defined. Limited sequencing of five LTRs (from a full del, a tandem arrangement and a solo LTR) indicates that the consensus termini are 5'TGT...ACA3', with del sequences flanked by a 5 bp tandem repeat. Thus del shares properties with retrotransposons such as the copia family of Drosophila and Ty of yeast. However with more than 13,000 copies per genome it seems del has been amplified more than is usual for such elements.
Mol Gen Genet 1989 Jan
PMID:An element with long terminal repeats and its variant arrangements in the genome of Lilium henryi. 271 Jan 4

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.
Mol Cell Biol 1988 Apr
PMID:Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo. 283 47

Centromeres most likely consist of DNA (CEN DNA) interacting with specific proteins. In Saccharomyces cerevisiae a clear picture has emerged of a 120 bp sequence that is characteristic of CEN DNA. We have investigated the 25 bp centromere DNA element (CDEIII) that represents the right part of a CEN DNA. We showed using a series of mutants generated in vitro that the right most triple A of the consensus sequence TGT.T.TG.. TTCCGAA.....AAA participates in the assembly of a functional centromere and that no further sequences to the right are needed. Distance changes between the centre dyad TTCCGAA and the triple A have two effects: Addition of one base pair leads to a reduction, and addition of two or four base pairs to a loss of centromere function implying a participation of the centre dyad and the triple A region in protein binding. Indeed, a synthetic oligonucleotide of 39 bp containing CDEIII shows specific protein binding.
Mol Gen Genet 1986 Nov
PMID:Mutations in the right boundary of Saccharomyces cerevisiae centromere 6 lead to nonfunctional or partially functional centromeres. 302 7

Five viable virus mutants were constructed with deletions near a 3' splice site located at nucleotide 2157 in the E3 transcription unit of adenovirus 2. The mutants were examined for splicing activity at the 2157 3' splice site in vivo by nuclease-gel analysis of steady-state cytoplasmic mRNA. Splicing was not prevented by an exon deletion (dl719) that leaves 16 5'-proximal exon nucleotides intact or by intron deletions that leave 34 (dl717, dl712) or 18 (dl716) 3'-proximal intron nucleotides intact. The sequences deleted in one of these intron mutants (dl716) include the putative branchpoint site used in lariat formation during splicing. Thus, a surrogate branchpoint site apparently can be used for splicing. Another intron mutant (dl714) has a deletion that leaves 15 3'-proximal intron nucleotides intact; remarkably, this deletion virtually abolished splicing, even though the deletion is only 3 nucleotides closer to the splice site than is the deletion in dl716 which splices normally. The three nucleotides deleted in dl714 that are retained by dl716 are the sequence TGT. The TGT sequence is located on the 5' boundary of the pyrimidine-rich region upstream of the nucleotide 2157 3' splice site. Such pyrimidine-rich regions are ubiquitous at 3' splice sites. Most likely, the TGT is required for splicing at the nucleotide 2157 3' splice site. The TGT may be important because of its specific sequence or because it forms the 5' boundary of the pyrimidine-rich region.
Mol Cell Biol 1985 Sep
PMID:Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2. 387 68

The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two-component sensor-regulator family. PilR was overproduced by fusing pilR to the gene for the maltose-binding protein (malE), yielding a MalE-PilR hybrid protein. The plasmid with the malE-pilR fusion, when introduced into a non-piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE-PilR protein was purified from Escherichia coli and used in a series of DNA-binding studies. A specific pilin promoter-binding activity of MalE-PilR was observed in a gel retardation assay. Subsequent DNase I footprinting analysis revealed a 40 bp PilR-binding site located at the -120 to -80 region, relative to the transcriptional start site of the pilin gene. This PilR-binding region consists of a nine-base sequence and three consensus sequences of 5'-(N)4-6C/GTGTC-3', in a tandem array in which the first 7-9 bp are bound by the PilR on the non-coding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds to sequences complementary to the two middle consensus sequences of the non-coding strand. A sequence similar to the NifA recognition site (5'-TGT-(N)11-ACA-3') is also found within the PilR-binding region. Deletion analysis and disruption of the individual consensus PilR-binding sequences by site-directed mutagenesis revealed that all four PilR-binding sites are absolutely required for the PilS/PilR-mediated pilin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Microbiol 1994 Dec
PMID:PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a cis-acting sequence upstream of the pilin gene promoter. 771 43

All mitochondrial tRNAs in the protozoan Leishmania are believed to be encoded in the nuclear genome and imported selectively into the mitochondria by an as yet unknown mechanism. Previously, we reported that two tRNAs whose genes are tightly linked were imported by mitochondria. In contrast, a tRNA encoded by a lone tRNA gene was not detectable in mitochondria. The lone tRNA gene had flanking sequences that were different from the linked genes. These studies implied a possible correlation between tRNA gene organization and gene flanking sequence, and selective tRNA import into mitochondria. Here, we report the identification of a cluster of 10 tRNA genes and show the distribution of the corresponding tRNAs in cytosolic and mitochondrial fractions. tRNA(leu)(CAG) and tRNA2(arg)(TCG) are abundant in the cytosol, but relatively scarce in mitochondria. Conversely, tRNA(ile)(TAT) and tRNA1(lys)(TTT) are abundant in mitochondria, but relatively scarce in the cytosol. tRNA(val)(TAC) and tRNA2(thr)(TGT) are barely detectable in either cellular compartment, while tRNA(gln)(TTG), tRNA1(arg)(ACG), tRNA(gly)(TCC), and tRNA(trp)(CCA) are detected in approximately equal levels in both compartments. Sequencing of the 2600 bp that comprise the tRNA gene cluster also encoding the genes for 5S RNA and URNAB RNA indicates that nucleotide composition, length, and location of genes within the cluster do not clearly correlate with import characteristics. The unexpected presence of the tRNA(trp)(CCA)-gene transcript in mitochondria is also reported. Evidence suggests that this tRNA may have unidentified base modifications at the anticodon triplet.
Mol Biochem Parasitol 1994 May
PMID:A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria. 793 26


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