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)

A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a beta-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate. In a model system using pSP65 or pGEM4 plasmids and transcripts, biotinylated RNA probes allowed detection of 5 pg of DNA in 10(6) pg of exogenous nucleic acids in 1000 min. Signals generated in the system depended on input target length. A nucleic acid target of 25 bases was still detectable in the assay. Human immunodeficiency virus type 1 (HIV-1) DNA was amplified in the polymerase chain reaction with Taq polymerase and a set of primers for the pol gene, one of which contained T7 RNA polymerase promoter sequences. A HIV-RNA probe of 326 bases was transcribed with T7 RNA polymerase using polymerase chain reaction (PCR) amplified DNA as a template. The RNA probe of 326 bases performed as well as a RNA probe of 2588 bases for detection of a DNA segment of 355 bp. For detection of dilutions of HIV-1 with PCR, a set of primers (outer set) was used for amplification of HIV-1 DNA. In a separate reaction a set of primers nested between the first set generated through PCR an amplified DNA fragment with the T7 promoter. This fragment was transcribed for the synthesis of a biotinylated RNA probe. This probe could then be reacted with material amplified with the outer set of primers. Ten copies of HIV-DNA could be detected with this procedure.
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PMID:Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids. 178 29

To define the RNA polymerase I promoter in the rDNA of Saccharomyces cerevisiae more precisely, we have constructed a series of 5'- and 3'-deletion mutants in a novel, plasmid-borne rDNA minigene, that also contains the transcriptional enhancer. Our data show that the Pol I promoter, in this context, extends from position -155 to +27, with 5'-deletions up to -134 and 3'-deletions up to -2 removing essential sequence information. To investigate the internal organization of the yeast Pol I promoter, linker scanning mutants were constructed, that traverse the Pol I promoter region and comprise between 5 and 12 clustered point mutations. Analysis of minigene transcription in yeast cells transformed with these plasmids demonstrates that the pol I promoter consists of three domains. Mutations in Domain I (from position -28 to +8) and Domain II (-70 to -51) drastically reduce promoter activity, whereas clustered point mutations in Domain III (starts at position -146 and presumably extends to position -76) appear to have less effect. Furthermore, the insertion of 4 nt between Domains I and II diminishes minigene transcription, indicating that the relative positions of these domains is essential.
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PMID:Linker scanning of the yeast RNA polymerase I promoter. 269 5

We have studied the sequence requirements for 3'-end formation of rDNA transcripts in a cell-free system and show that the generation of correct ends of mouse pre-rRNA is brought about by a two-step process that involves a bona fide termination reaction, followed by a specific trimming of the primary transcript by 10 nucleotides. We show that termination of mouse ribosomal gene transcription by RNA polymerase I (pol I) takes place in front of an 18-bp DNA sequence element (the 'Sal box'), which was previously shown to function as termination signal. Termination of pol I transcription occurs at a fixed distance (11 bp) upstream of the Sal box, independent of the sequence of adjacent gene regions. The processing reaction, however, is strongly influenced by sequences flanking the termination signal at the 5' site. Substitution of a cluster of T residues by guanines within the region of 3'-end formation abolishes the 3'-terminal trimming of the primary transcript. Interestingly, this 3'-terminal processing event, which can be uncoupled from the termination reaction, requires both a correct 3' end and specific sequences in the 3'-terminal region of the primary transcript. Read-through transcripts generated in the extract system or by SP6 RNA polymerase are no substrate for the processing nuclease(s). Because the termination and processing activity can be separated chromatographically, the nucleolytic activity does not reside in TTF-I, the factor that binds to the Sal box and directs transcription termination.
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PMID:3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction. 271 50

Nucleotide sequence analysis of a cDNA clone corresponding to the XBL-I open reading-frame of bovine leukemia virus (BLV) revealed that the AUG initiation codon was located 44 bases downstream from that of the env gene and was part of the p34x mRNA splice donor. . .ATGG/GTAA at the end of the pol gene sequence. RNA from this clone was synthesized in vitro by the SP6 RNA polymerase and translated into a 34,000 mol wt protein in rabbit reticulocyte lysates. The protein (p34x) is recognized in Western blots by most sera of BLV-infected sheep and tumor-bearing cattle, by an anti-synthetic peptide rabbit serum, and by the serum of a rabbit immunized by XBL-I RNA programmed reticulocyte lysates. Both sera react with a 34,000 mol wt protein present in nuclei of BLV-infected cells.
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PMID:Expression of a cDNA clone corresponding to the long open reading frame (XBL-I) of the bovine leukemia virus. 282 Jan 39

RNA polymerase III (pol III) transcribes the highly repeated murine B2 elements. We showed previously that the B2 RNAs are induced 4-fold in normal growing cells and 20-fold in simian virus 40-transformed cells relative to the levels in normal confluent cells. By employing chromatin as a template in a partially purified pol III transcription system, we now demonstrate that the augmented expression results from the formation of pol III transcription complexes on previously inactive B2 genes. Extracts prepared from normal growing cells and transformed cells transcribed cloned pol III templates 5-fold more efficiently than extracts from normal confluent cells. This increase was attributed to 5-fold greater levels of factor IIIC; the levels of pol III and factor IIIB were the same in all extracts. We discuss how the levels of IIIC and differing accessibility of this factor to repressed B2 genes mediate the formation of pol III transcription complexes in normal growing and transformed cells.
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PMID:Enhanced B2 transcription in simian virus 40-transformed cells is mediated through the formation of RNA polymerase III transcription complexes on previously inactive genes. 284 94

The structural requirements for 3' end formation of mouse pre-rRNA have been studied. Three sequence elements are shown to be required for accurate and efficient transcription termination by RNA polymerase I (pol I) assayed both in a cell-free transcription system and in vivo after transfection of rDNA minigene constructs into 3T6 cells. The essential termination signal is the previously identified 18-bp conserved element (AGGTCGACCAGATTANTCCG) that contains a SalI restriction site. This sequence motif (the 'Sal box') interacts with a specific nuclear protein that directs transcription termination. Here we demonstrate that the 'Sal box' sequence motif is sufficient for termination of pol I transcripts and the release of the nascent RNA chains from the template. However, in addition to this termination signal, pyrimidine-rich sequences flanking the box at the 5' and 3' side play a role in the efficient and correct formation of authentic pre-rRNA termini. Downstream sequences contribute to the efficiency of the termination reaction, whereas the position of 3' end formation (i.e. 21 bp upstream of the 'Sal box') is affected by 5' flanking regions. These flanking regions are recognized by at least two different nuclear factors which specifically bind to DNA sequences located upstream and downstream of the 'Sal box'.
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PMID:The mouse ribosomal gene terminator consists of three functionally separable sequence elements. 290 Jul 60

We have isolated the genes encoding the largest subunit of all three classes of RNA polymerase from Trypanosoma brucei. While the pol II largest subunit is encoded by a single gene in all organisms examined to date, trypanosomes contain two copies of the gene. Both genes are expressed in the procyclic and bloodstream stages of the trypanosome life cycle. The two pol II genes differ from one another in their coding sequences by 21 silent substitutions and 4 amino acid substitutions. In the core part of the large subunit, the predicted polypeptides are similar to other eukaryotic RNA polymerases. Both trypanosome pol II polypeptides, like those of other eukaryotes, also have a unique C-terminal extension. However, this domain in the trypanosome polypeptides, unlike those of other eukaryotes, is not a tandemly repeated heptapeptide sequence.
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PMID:In trypanosomes the homolog of the largest subunit of RNA polymerase II is encoded by two genes and has a highly unusual C-terminal domain structure. 292 50

Replication of plasmids that depend on the 245-base-pair origin of the Escherichia coli chromosome (oriC) requires many purified proteins that (i) direct initiation to oriC (e.g., dnaA protein), (ii) influence initiations elsewhere (e.g., auxiliary proteins), and (iii) prime and extend DNA chains (e.g., priming and synthesis proteins). For the RNA priming and initiation of new DNA chains, the requirements for both primase and RNA polymerase (RNA pol) [Kaguni, J. M. & Kornberg, A. (1984) Cell 38, 183-190] have been further analyzed. Depending on the levels of auxiliary proteins (topoisomerase I and protein HU), three priming systems can operate: primase alone, RNA pol alone, or both combined. At low levels of auxiliary proteins, primase alone sustains an effective priming system. At higher levels, primase action is blocked, but RNA pol alone can initiate replication, albeit feebly; at these high levels of auxiliary proteins, primase and RNA pol act synergistically. When RNA pol is stalled by an inhibitor or lack of a ribonucleoside triphosphate, primase action is also inhibited. Based on these and other data [van der Ende, A., Baker, T. A., Ogawa, T. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, in press], RNA pol can counteract inhibition by auxiliary proteins and thus activate the origin for the priming by primase of the leading strand of the replication fork.
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PMID:Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: contributions of RNA polymerase and primase. 298 33

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.
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PMID:Induction of specific transcription by RNA polymerase III in transformed cells. 302 60

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
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PMID:Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter. 318 28


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