Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.
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PMID:Myc protein structure: localization of DNA-binding and protein dimerization domains. 199 48

The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase II transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCl. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.
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PMID:Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. 201 92

Genes of higher eucaryotic cells are considered to show only a limited response to nutritional stress. Here we show, however, that omission of a single essential amino acid from the medium caused a marked rise in the mRNA levels of c-myc, c-jun, junB and c-fos oncogenes and ornithine decarboxylase (ODC) in CHO cells. There was no general accumulation of mRNAs in amino acid-starved cells, since the gamma-actin, beta-tubulin, protein kinase C, RNA polymerase II, and glyceraldehyde-3-phosphate dehydrogenase mRNAs and the total poly(A)+ mRNA were not increased. The levels of c-myc, ODC, and c-jun mRNAs were elevated more by amino acid starvation than by inhibition of protein synthesis with cycloheximide, which is known to increase the levels of these mRNAs. Importantly, however, cycloheximide present during amino acid starvation reduced the rise in the levels of the mRNAs down to the level obtained with cycloheximide alone. This implies that protein synthesis is required for the accumulation of c-myc, ODC, and c-jun mRNAs in amino acid-deprived cells. The junB and c-fos mRNAs, instead, were increased to the same extent or less by amino acid starvation than by cycloheximide treatment. The accumulation of the c-myc mRNA in amino acid-starved cells was due to both stabilization of the mRNA and increase of its transcription. The rise in the c-jun mRNA level seemed to be caused merely by stabilization of the mRNA. Further, despite the inhibition of general protein synthesis, amino acid starvation led to an increase in the synthesis of c-myc polypeptide. The results suggest that mammalian cells have a specific mechanism for registering shortages of amino acids in order to make adjustments compatible with cellular growth.
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PMID:Deprivation of a single amino acid induces protein synthesis-dependent increases in c-jun, c-myc, and ornithine decarboxylase mRNAs in Chinese hamster ovary cells. 212 33

Eukaryotic RNA polymerase II recognizes certain DNA sequences as effective signals for transcription termination in vitro. Previously, we have shown that such termination occurs within T-rich sequences; however, not all T runs stop the enzyme nor is the efficiency of termination correlated with the length of the T run. Here we have investigated the sequence elements that signal transcription termination by purified RNA polymerase II. We have examined terminators located within introns of the human histone H3.3 gene and the human c-myc gene. Deletion analysis of the H3.3 termination region indicates that the sequences between -6 and +24 relative to the strongest termination site are sufficient to cause transcription termination. The minimal termination signal at this site has been localized to the sequence TTTTTTTC-CCTTTTTT in the nontranscribed strand. A similar but nonidentical sequence has been defined for the c-myc termination site. Since RNA polymerase II terminates transcription only within the first run of T residues in these sequences, at least part of the termination signal lies in downstream nontranscribed DNA sequences. Restriction fragment mobility analysis indicates that the H3.3 termination region contains a bend in the DNA helix. Oligonucleotides containing the minimal termination signals also cause restriction fragments to migrate with anomalous mobility. A region of the SV40 genome containing a previously characterized bend also causes RNA polymerase II to terminate transcription. We suggest that a structural element causing a bend in the DNA helix may be part of the signal for transcription termination by purified RNA polymerase II.
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PMID:Analysis of the signals for transcription termination by purified RNA polymerase II. 215 81

Human cells contain a nuclear protein interacting with Alu repeats, and this protein seems to recognize a conserved sequence motif, GGAGGC, present within the RNA polymerase III promoter and within the SV40 T-antigen-dependent ARS-like element. To study the potential functional role of this element, we have inserted the sequence into a chloramphenicolacetyltransferase (CAT) expression vector with a SV40 promoter and enhancer element from the up-stream region of the human c-myc gene, and transfected HeLa cells with the resulting plasmid. Analysis of expression by the CAT assay indicates that the Alu-derived sequence supresses transcription of the CAT gene driven by the c-myc enhancer/SV40 promoter. The Alu-derived sequence also inhibits ARS activity of the c-myc enhancer. The data allow the explanation of the transcriptional inactivity of Alu repeats in HeLa cells, and suggest the existence of a negative control of Alu transcription.
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PMID:Transcription and replication silencer element is present within conserved region of human Alu repeats interacting with nuclear protein. 215 7

A new affinity chromatographic procedure for the separation of transcriptionally active nucleosomes has been used to study the changes that take place in chromatin structure along the c-fos and c-myc genes when RNA synthesis is inhibited. Mercury-affinity chromatography separates the sulfhydryl-reactive nucleosomes of transcriptionally active genes from the compactly beaded, non-reactive nucleosomes of transcriptionally inert DNA sequences. The new procedure also discriminates between nucleosomes that have "unfolded" to reveal the previously shielded SH groups of histone H3 and nucleosomes that bind to the mercury column because of their association with thiol-containing non-histone proteins located in the transcription unit. Both classes of Hg-bound nucleosomes contain the c-fos and c-myc sequences, but only when they are being transcribed. We compared the effects of alpha-amanitin and actinomycin D on the transcription of c-fos and c-myc with the effects of each inhibitor on the distribution of the corresponding oncogenic DNA sequences in the chromatographically separated nucleosome fractions. It was found that the inhibition of RNA polymerase II by alpha-amanitin (added at the peaks of c-fos or c-myc expression in serum-stimulated BALB/c 3T3 cells) resulted in a rapid loss of affinity of the oncogene-containing nucleosomes for the mercury column. There was no corresponding effect on the mercury-binding properties of nucleosomes containing 28 S ribosomal gene sequences, which continue to be transcribed by amanitin-resistant RNA polymerase I. Therefore, the binding of the c-fos and c-myc nucleosomes to the mercury column seems to depend upon reversible structural changes associated with their transcription. Surprisingly, there was no corresponding loss of affinity of the c-fos and c-myc nucleosomes for the mercury column when actinomycin D was employed to inhibit RNA synthesis, despite the fact that transcription of both genes had been arrested abruptly. Measurements of [3H]actinomycin D binding show its preferential intercalation into the transcriptionally active nucleosomes. We suggest that the intercalation of actinomycin D into the DNA of active nucleosomes can lock the transcription complex into an "unfolded" but potentially active configuration. This was confirmed by run-off transcription assays showing a restoration of c-fos and c-myc RNA synthesis when actinomycin D was displaced by proflavine.
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PMID:Reversible and irreversible changes in nucleosome structure along the c-fos and c-myc oncogenes following inhibition of transcription. 232 30

We have investigated a number of genes for possible overexpression upon induction of transformation, using mouse cells transformed with a temperature-sensitive mutant of simian virus 40 [SV40Ts(A)-transformed BALB-3T3, line A255], which express the transformed phenotype at 33 degrees C and suppress it as 39 degrees C. We examined both RNA polymerase II transcripts belonging to selected cellular oncogenes and RNA polymerase III transcripts of the B2 class. The selection was predicated on the basis of previous reports of an overexpression of these genes in transformed cell lines or tumors. Among the oncogenes, only cellular myc mRNA was found in increased amounts after transfer to the permissive temperature. However, nuclear run-on transcription assays indicated accelerated transcription rates for both c-myc and c-ras, with 2-3-fold and 4-5-fold increases at 2 and 6 hr post-induction, respectively. This acceleration was preceded by a 16-fold increase in the transcription rate of mouse B2 genes occurring within 30 min of the transfer to 33 degrees C. The sizeable and immediate burst in the rate of B2 transcription following the induction shift and the temporal order of activation of c-myc and c-ras are suggestive of a causal progression leading to full expression of the transformed phenotype.
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PMID:Increased rates of specific RNA polymerase III transcription precede overexpression of cellular oncogenes upon induction of transformation. 247 9

Two distinct Xenopus c-myc cDNA clones have been characterized from an oocyte cDNA library. This allowed a comparison of the c-myc protein sequence across the vertebrate phylum to be made and prominent conservations to be identified. The majority of the sequence differences between the two Xenopus c-myc cDNAs are in the 5' and 3' untranslated regions. Sequence-specific oligonucleotide probes from the 5' untranslated region were used to demonstrate the differential expression of the two c-myc mRNAs during development. One of the mRNAs corresponds to the Xenopus c-myc gene previously reported expressed as a stable maternal mRNA uncoupled from cell division during oogenesis (c-myc I). It is the major mRNA species expressed during oogenesis and is expressed again from the zygotic genome in post-gastrula embryos. In contrast, the second c-myc mRNA (c-myc II) is expressed only from the maternal genome during oogenesis. Primer extension experiments show that in the oocyte the transcriptional initiation sites for c-myc I and c-myc II are at different distances from the translational start site. The 'oocyte-specific' and 'somatic-type' developmental regulation of c-myc is reminiscent of polymerase III 5S RNA gene expression in Xenopus, and may provide new insights into the developmental regulation of genes transcribed by RNA polymerase II.
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PMID:Differential expression of two Xenopus c-myc proto-oncogenes during development. 268 81

We have investigated the factors that permit a gene normally transcribed by RNA polymerase II to be transcribed by RNA polymerase III. It was shown previously that the human c-myc gene could be transcribed in vitro and in Xenopus oocytes by both alpha-amanitin-sensitive and alpha-amanitin-resistant polymerases, probably corresponding to polymerase II and polymerase III. We confirmed this observation in microinjected oocytes and showed that the alpha-amanitin-resistant transcription of c-myc was competed by known polymerase III genes. Polymerase III transcription of c-myc was very inefficient compared to other polymerase III genes, however, and was observed only when large amounts of template DNA were injected. At lower DNA concentrations the gene was transcribed, exclusively by polymerase II. In contrast, the adenovirus major late promoter was not transcribed by polymerase III. The 5' ends of polymerase III RNAs were almost indistinguishable from those of polymerase II RNAs initiating at the P1 and P2 promoters of the human and mouse c-myc genes. Furthermore, point mutations in the TATA box of the human P2 promoter greatly reduced polymerase III activity. At this promoter, therefore, polymerase II and polymerase III recognize a common element, the TATA box, which probably plays an important role in specifying the start site of transcription for both polymerases. We suggest that the highly accurate though inefficient mimicry of polymerase II by polymerase III at the c-myc promoters reflects the common evolutionary origin of these two enzymes.
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PMID:Accurate, TATA box-dependent polymerase III transcription from promoters of the c-myc gene in injected Xenopus oocytes. 279 59

We have used the Xenopus oocyte injection system to investigate the sequence requirements of premature termination of transcription within the human c-myc gene. We show that in the oocyte, truncated RNAs are produced by RNA polymerase II with 5' ends at the P1 and P2 promoters and 3' ends at two T stretches (sites I and II) near the exon 1/intron 1 junction. The location of these 3' ends is consistent with the site of the block to c-myc transcription identified by nuclear runoff assays in human cells and confirmed in dissected nuclei of injected oocytes. Evidence is presented that transcriptional termination rather than RNA processing produces these short c-myc RNAs. Deletion analysis of site I reveals that sequences upstream of the T stretch determine the site of 3' end formation, and that the stretch of T's on the sense DNA strand is not required for termination. The sequences specifying termination reside within a 95 base region located -130 to -35 relative to the exon 1/intron 1 boundary. The termination activity of these sequences is orientation-dependent and functions downstream of the HSV-TK promoter.
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PMID:Sequence requirements for premature termination of transcription in the human c-myc gene. 283 65


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