Purification of Drosophila nucleosome remodeling factor.
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Animals↗
Biomedical subjects
V Ossipow
Publications and source records attributed to V Ossipow.
Heterogeneous nuclear RNA-ribonucleoprotein F binds to DNA via an oligo(dG)-motif and is associated with RNA polymerase II.
BACKGROUND: The heterogeneous nuclear ribonucleoprotein F (hnRNP-F) is one of the constituents of the splicing-related hnRNP complex. Recent studies suggest that pre-mRNA modification and splicing factors are associated with transcriptional initiation factors and RNA polymerase II (RNA pol II) at a promoter, implying that pre-mRNA-engaged factors might be associated with a promoter. RESULTS: We isolated a cDNA of rat hnRNP-F and expressed the recombinant protein. HnRNP-F selectively bound to oligo(dG) in constructions with other oligohomonucleotides. The ssDNA of an SV40 promoter sequence having the GC-boxes was bound to hnRNP-F, while hnRNP-F bound to the (G/C)-stretch as dsDNA. Consequently, hnRNP-F was designated as an oligo(dG)-binding protein. None of the RNA-binding domains (RBDs) in hnRNP-F were critical for the oligo(dG)-binding. In contrast, the GY-rich region that exists between RBD-II and -III exhibited an oligo(dG)-binding ability. We suggest that the GY-rich region is a novel DNA-binding motif. HnRNP-F was shown to contain two types of binding motifs for RNA and DNA. Additionally, we found that hnRNP-F was co-precipitated with RNA pol II. Interestingly, the RNA pol II holoenzyme also contained hnRNP-F. The tissue distribution profile of hnRNP-F was similar to that of both RNA pol II and TBP. CONCLUSION: The above results suggest that hnRNP-F is associated with a transcriptional initiation apparatus that includes RNA pol II. The DNA-binding ability of hnRNP-F might facilitate the entry of pre-mRNA modification/splicing factors at a promoter.
Amino Acid Sequence↗
An RNA polymerase II complex containing all essential initiation factors binds to the activation domain of PAR leucine zipper transcription factor thyroid embryonic factor.
Transcription initiation of protein-encoding genes involves the assembly of RNA polymerase II and a number of general transcription factors at the promoter. A mammalian RNA polymerase II complex containing all of the components required for promoter-specific transcription initiation can be isolated by immunopurification with a monoclonal antibody directed against the cyclin-dependent kinase CDK7, a subunit of the general transcription factor TFIIH. In vitro transcription by this immunopurified RNA polymerase II complex is effectively stimulated by thyroid embryonic factor (TEF), a basic leucine zipper transcription factor. Thus, the RNA polymerase II complex must also contain components required for activated transcription that interact with the transactivation domain of TEF. This conjecture was verified by affinity selection experiments in which the TEF transcription activation domain was used as a bait. Indeed, an RNA polymerase II complex containing all of the accessory proteins required for transcription initiation can be enriched by its affinity to recombinant proteins containing the TEF transactivation domain. These results are compatible with a mechanism by which TEF can recruit an RNA polymerase II holoenzyme to the promoter in a single step.
Animals↗
Inorganic pyrophosphatase is a component of the Drosophila nucleosome remodeling factor complex.
The Drosophila nucleosome remodeling factor (NURF) is a protein complex consisting of four polypeptides that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The 140-kD NURF subunit, imitation switch (ISWI), is related to the SWI2/SNF2 ATPase. Another subunit, NURF-55, is a 55-kD WD repeat protein homologous to the human retinoblastoma-associated protein RbAp48. Here, we report the cloning and characterization of the smallest (38 kD) component of NURF. NURF-38 is strikingly homologous to known inorganic pyrophosphatases. Both recombinant NURF-38 alone and the purified NURF complex are shown to have inorganic pyrophosphatase activity. Inhibition of the pyrophosphatase activity of NURF with sodium fluoride has no significant effect on chromatin remodeling, indicating that these two activities may be biochemically uncoupled. Our results suggest that NURF-38 may serve a structural or regulatory role in the complex. Alternatively, because accumulation of unhydrolyzed pyrophosphate during nucleotide incorporation inhibits polymerization, NURF may also have been adapted to deliver pyrophosphatase to chromatin to assist in replication or transcription by efficient removal of the inhibitory metabolite.
Amino Acid Sequence↗
ATP-dependent remodeling of chromatin.
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Adenosine Triphosphatases↗
The two PAR leucine zipper proteins, TEF and DBP, display similar circadian and tissue-specific expression, but have different target promoter preferences.
The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50-fold and 160-fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co-transfection experiments, these two proteins exhibit different activation potentials for two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF-DBP fusion protein, carrying N-terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH-CAT reporter gene as strongly as wild-type DBP. Our results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP.
Animals↗
A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation.
The protein kinase MO15/CDK7 has recently been shown to be associated with the general transcription factor TFIIH and to be capable of phosphorylating the RNA polymerase II carboxy-terminal domain. Here, we show that a monoclonal MO15/CDK7 antibody coimmunoprecipitates, from a rat liver nuclear extract, all components of the RNA polymerase II transcription apparatus required for initiation at the albumin and adenovirus major late promoters. The immunoprecipitate includes RNA polymerase II, TFIID, TFIIB, TFIIH, TFIIF, and TFIIE, but is devoid of transcriptional activator proteins, such as HNF1, HNF4, and C/EBP alpha. The finding of an autonomously initiating RNA polymerase II holoenzyme in mammalian cells suggests conceptual similarities between transcription initiation in prokaryotes and eukaryotes.
Animals↗
A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis.
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Animals↗
CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials.
The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one. Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved in vertebrate evolution.
Affinity Labels↗
The role of the transcriptional activator protein DBP in circadian liver gene expression.
DBP, a liver-enriched transcriptional activator protein of the leucine zipper protein family, accumulates according to a very strong circadian rhythm (amplitude approx. 1000-fold). In rat parenchymal hepatocytes, the protein is barely detectable during the morning hours. At about 2 p.m., DBP levels begin to rise, reach maximal levels at 8 p.m. and decline sharply during the night. This rhythm is free-running: it persists with regard to both its amplitude and phase in the absence of external time cues, such as daily dark/light switches. Also, fasting of rats for several days influences neither the amplitude nor the phase of circadian DBP expression. Since the levels of DBP mRNA and nascent transcripts also oscillate with a strong amplitude, circadian DBP expression is transcriptionally controlled. While DBP mRNA fluctuates with a similar phase and amplitude in most tissues examined, DBP protein accumulates to high concentrations only in liver nuclei. Hence, at least in nonhepatic tissues, cyclic DBP transcription is unlikely to be controlled by a positive and/or negative feedback mechanism involving DBP itself. More likely, the circadian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glucocorticoid hormones in establishing the DBP cycle. Two genes whose mRNAs and protein products accumulate according to a strong circadian rhythm with a phase compatible with regulation by DBP encode enzymes with key functions in cholesterol metabolism: HMG-coA reductase is the rate-limiting enzyme in cholesterol synthesis; cholesterol 7-alpha hydroxylase performs the rate-limiting step in the conversion of cholesterol to bile acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗