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)

The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments. 301

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.
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PMID:Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences. 301 46

To investigate the chromatin surrounding an active gene, we have determined the distribution of RNA polymerase molecules, the intactness of nucleosomal structure, and the subnuclear compartmentalization along 15 kilobase pairs (kb) of the mouse kappa immunoglobulin locus of MPC-11 plasmacytoma cells. Hybridization of in vitro nuclear transcripts to probes specific for the template strand reveals that transcription terminates within the region between 1.1 and 2.3 kb downstream from the poly(A) addition site. Ten different short sequences (8-13 base pairs) reside within 460 base pairs of this termination region that exhibit homology with sequences found in the termination regions of mouse beta-globin and chicken ovalbumin genes. Transcription of the nontemplate strand occurs on either side of this termination region. We find that both within the transcription unit and 6.5 kb downstream of the termination region of the kappa gene, the canonical nucleosomal structure is perturbed, the chromatin exhibits pronounced insolubility, and the nucleosomes liberated by micrococcal nuclease appear to lack histone H1. The insolubility is characterized by interactions that are disrupted by 0.3 to 0.6 M NaCl treatment. We conclude that the active chromatin phenotype spreads a considerable distance along the kappa locus, well beyond the region of transcription termination.
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PMID:Transcription termination and chromatin structure of the active immunoglobulin kappa gene locus. 308 10

We examined the intramolecular effect of altered cap structures on translation efficiency of artificial beta-globin mRNAs. For these studies, synthetic dinucleotides of the form X(5')ppp(5')G [X = 7-methyl guanosine (m7G), 2,7-dimethyl guanosine (m2(2,7)G) or 2,2,7-trimethyl guanosine (m3(2,2,7)G)], were transcriptionally incorporated into mRNAs, containing rabbit beta-globin coding sequences, using T7 RNA polymerase and a beta-globin cDNA template. These synthetic mRNAs were assayed in reticulocyte lysate for activity relative to m7G-capped mRNA. m2(2,7)G-Capped mRNA was found to be 1.5-fold more active than m7G-capped mRNA. Messenger RNA capped with m3(2,2,7)G was less active with activity of 0.24 relative to its m7G-capped counterpart (activity = 1.0). These data suggest that m7G-capped mRNAs become more active as translation templates after addition of a single N2 methyl moiety, which is especially pertinent to gene expression in togaviridae. The latter are observed to synthesize m2(2,7)G and m3(2,2,7)G-capped mRNAs in addition to m7G-capped templates during the course of infection in animal cells.
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PMID:Beta-globin mRNAs capped with m7G, m2.7(2)G or m2.2.7(3)G differ in intrinsic translation efficiency. 317 38

Oligonucleotide-limited transcription has been used to prepare a series of transcripts which allowed the positions of termination by T7 RNA polymerase to be characterized. The same technique was used to prepare a set of transcripts from a rabbit beta-globin gene that extend in intervals of two nucleotides from the 3' splice site of IVS-1 into the second exon. Splicing efficiency in a HeLa cell nuclear extract decreased with decreasing length of the 3' exon, although both steps of the splicing reaction could still be detected with as few as four nucleotides in this exon. No evidence was found for a lower limit to the length of the 3' exon below which splicing would not take place. With longer substrates, the rate of the second step of splicing was increased substantially.
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PMID:The dependence of splicing efficiency on the length of 3' exon. 342 5

The initiation of transcription by RNA polymerase II in isolated murine erythroleukemia cell nuclei was investigated by isolating newly synthesized gamma-thio (gamma-S-)-triphosphate-labeled transcripts by Hg-agarose chromatography. The 5' terminus of transcripts initiated in vitro with [gamma-35S]ATP or [gamma-35S]GTP was identified as the thiotetraphosphate in alkaline hydrolysis products from Hg-agarose-selected RNA. Additional control experiments analyzing the nuclear transcription of two well characterized tRNA genes showed that each gene was initiated with the proper triphosphate, either gamma-S-ATP or gamma-S-GTP, indicating little, if any, exchange of the gamma-S-labeled substrate to the other triphosphates. As determined by S1 mapping, newly synthesized beta-globin gene transcripts initiate only with gamma-S-ATP. Their 5'-terminus is located at the cap site, and their synthesis is inhibited by 1 microgram alpha-amanitin/ml. In reactions containing gamma-S-ATP but not gamma-S-GTP, several additional initiation sites are observed that are located in the 5'-flanking region. We conclude that RNA polymerase II can initiate transcription at the cap site in isolated nuclei.
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PMID:Accurate in vitro initiation of beta-globin gene transcription in induced Friend-cell nuclei. 346 70

The response of isolated rat liver and murine erythroleukemia nuclei to phospholipid liposomes has been monitored with different techniques, by studying the endogenous RNA synthesis, the release of transcripts in the medium, the pattern of acid-extractable nuclear proteins and the ultra-structural morphology. Total transcription in rat liver and beta-globin mRNA synthesis in MEL nuclei are increased by PS and reduced by PC. These changes of RNA polymerase activity, and the transport of RNAs from nucleus as well as the nuclear protein changes, correlate with structural transitions which occur in both types of nuclei, consisting of euchromatization with loss of RNP particles in the case of PS and opposite effects with PC. The significance of these modifications in relationship to the possible involvement of phospholipids in the control of gene expression is discussed.
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PMID:Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei. 346 78

Sequence elements within the mouse beta maj-globin transcription unit required for efficient termination of transcription by RNA polymerase II have been delineated. To facilitate nascent-chain analysis of termination, the DNA segment in which transcription ceases was introduced into the adenovirus chromosome within its E1A transcription unit. Two beta-globin DNA elements were required to effect efficient termination: an upstream sequence that includes two poly(A) addition signals and a downstream region previously shown to be where RNA synthesis stops. The role of poly(A) addition in termination was established by introduction of several single base pair substitutions into the AATAAA polyadenylylation motifs. These mutations inhibited both polyadenylylation and termination within the beta-globin DNA segment. Therefore, poly(A) addition appears to be a prerequisite for efficient termination.
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PMID:A poly(A) addition site and a downstream termination region are required for efficient cessation of transcription by RNA polymerase II in the mouse beta maj-globin gene. 347 94

We have identified and mapped several DNA sequences within a human histone gene (H3.3) at which in-vitro transcription by highly purified RNA polymerase II is efficiently terminated. Since transcription in our system involves only RNA polymerase II acting on a linear DNA template, these sequences contain "intrinsic" termination signals recognized by the polymerase protein itself. The existence of such signals within a gene suggests that efficient antitermination systems probably exist for mammalian transcription units. Alternatively, there could be a high frequency of premature transcription termination, or "polarity" for genes such as H3.3. Intrinsic transcription termination sites in H3.3 are located in sequences of consecutive thymidylate residues (5 to 8 nucleotides) on the non-transcribed DNA strand (T-runs), from which it is likely that such T-runs are elements of the intrinsic termination signal for RNA polymerase II. However, transcription proceeds without significant termination through many similar T-runs, from which it follows that these intrinsic termination signals include other elements. Since transcription is also terminated efficiently at these sites when the transcript remains bound along its full length as a DNA-RNA hybrid, it is unlikely that formation of specific RNA secondary structures in the transcript is a general element of the intrinsic termination signal. Although DNA sequences downstream from the coding portion of the mouse beta-globin gene have been implicated as sites of transcription termination in vivo, these regions do not contain strong intrinsic termination signals, and transcription in vitro proceeds through these regions almost undiminished. Transcriptional termination in this region in vivo may depend on the presence of termination factors or other intracellular elements, and there may be multiple classes of DNA signals that control eukaryotic termination.
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PMID:Identification of intrinsic termination sites in vitro for RNA polymerase II within eukaryotic gene sequences. 365 48

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and alpha-genes. In contrast, the the simian virus 40 early genes, the beta-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-II factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.
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PMID:Identification of two factors required for transcription of the ovalbumin gene. 379 2


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