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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The initiation site of the major transcript of rice tungro bacilliform virus (RTBV) has been located by mapping the 5' end of the RNA to nucleotides 7404 and 7405 of the RTBV genome using an RNase protection method. This was confirmed by the 5' RACE PCR procedure which mapped the 5' end of the RNA to nucleotide 7405. These results are consistent with data from our analysis of the strong-stop DNA of RTBV. A eukaryotic RNA polymerase II promoter sequence (TATATAA) was located at nucleotide 7373 of RTBV genome which is 31-32 nucleotides upstream from the proposed initiation site of the RTBV transcript.
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PMID:Mapping the 5'-terminus of rice tungro bacilliform viral genomic RNA. 821 81

RNA chain elongation by RNA polymerase is a dynamic process. Techniques that allow the isolation of active elongation complexes have enabled investigators to describe individual steps in the polymerization of RNA chains. This article will describe recent studies of elongation by RNA polymerase II (pol II). At least four types of blockage to chain elongation can be overcome by elongation factor SII: (a) naturally occurring "arrest" sequences, (b) DNA-bound protein, (c) drugs bound in the DNA minor groove, and (d) chain-terminating substrates incorporated into the RNA chain. SII binds to RNA polymerase II and stimulates a ribonuclease activity that shortens nascent transcripts from their 3' ends. This RNA cleavage is required for chain elongation from some template positions. As a result, the pol II elongation complex can repeatedly shorten and reextend the nascent RNA chain in a process we refer to as cleavage-resynthesis. Hence, assembly of large RNAs does not necessarily proceed in a direct manner. The ability to shorten and reextend nascent RNAs means that a transcription impediment through which only half the enzyme molecules can proceed per encounter, can be overcome by 99% of the molecules after six iterations of cleavage-resynthesis. Surprisingly, the boundaries of the elongation complex do not move upstream after RNA cleavage. The physico-chemical alterations in the elongation complex that accompany RNA cleavage and permit renewed chain elongation are not yet understood.
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PMID:Transcription elongation by RNA polymerase II: mechanism of SII activation. 831 68

Osteoblast-enriched (Ob) cultures isolated from fetal rat bone synthesize insulin-like growth factor-I (IGF-I), which functions as a locally acting growth and differentiation factor in the skeleton. Consistent with prior studies demonstrating that IGF-I production is enhanced in bone by agents that induce cAMP, prostaglandin E2 (PGE2) stimulates both cAMP synthesis and IGF-I mRNA in Ob cells. However, little is known about how cAMP regulates IGF-I expression in this or any other cell system. In rat tissues, multiple mechanisms influence levels of IGF-I mRNA, including transcription from two promoters, differential RNA splicing and stability, and alternative RNA polyadenylation. To determine how cAMP influences IGF-I gene expression in Ob cultures, we examined the responses of these cells to treatment with PGE2. PGE2 rapidly enhanced the accumulation of both large and small IGF-I transcripts, with increases in IGF-I mRNA detected within 2 h of treatment and persisting for 24 h. Analysis of precursor RNA by a highly specific and sensitive ribonuclease protection assay demonstrated a rise in nascent IGF-I mRNA within 30 min of exposure to PGE2, with a peak stimulation of 4-fold above control levels seen by 2 h and levels remaining elevated for up to 24 h. IGF-I transcripts in Ob cells were directed only by promoter 1, the more 5' of the two rat IGF-I gene promoters. As additionally assessed using the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, PGE2 treatment had little effect on IGF-I mRNA stability. In aggregate, these studies show that in fetal rat Ob cultures, PGE2 enhances IGF-I gene expression primarily through transcriptional mechanisms that are limited to a single IGF-I gene promoter. Ob cells, therefore, may be an excellent model for determining how cAMP regulates IGF-I gene transcription.
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PMID:Prostaglandin E2 rapidly stimulates insulin-like growth factor-I gene expression in primary rat osteoblast cultures: evidence for transcriptional control. 839 6

Elongation factor SII is required to increase the efficiency of transcription by RNA polymerase II through intrinsic arrest sites. RNA polymerase II ternary complexes exhibit a ribonuclease activity in the presence of SII, truncating nascent transcripts in a 3'-->5' direction. We show here that transcript cleavage is an obligatory step in re-establishing the elongation competency of complexes that have become blocked in elongation at an intrinsic arrest site. SII-facilitated transcript cleavage by these arrested complexes released 7-14 nucleotide RNA fragments. In contrast, SII-facilitated transcript cleavage by elongation competent complexes, which are stalled because of the absence of a nucleoside triphosphate from the reaction mixture, occurred primarily in dinucleotide increments. We can partially recreate the arrested phenotype and the preference for the large cleavage increment by stalling ternary complexes such that the 3'-end of the transcript contains consecutive U residues, which mimics the sequence of the 3'-ends of transcripts in arrested complexes.
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PMID:The increment of SII-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes. 850 21

We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.
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PMID:Three functional classes of transcriptional activation domain. 862 70

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.
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PMID:Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest. 869 22

Elongation factor SII (also known as TFIIS) is an RNA polymerase II binding protein that allows bypass of template arrest sites by activating a nascent RNA cleavage reaction. Here we show that SII contacts the 3'-end of nascent RNA within an RNA polymerase II elongation complex as detected by photoaffinity labeling. Photocross-linking was dependent upon the presence of SII, incorporation of 4-thio-UMP into RNA, and irradiation and was sensitive to treatment by RNase and proteinase. A transcriptionally active mutant of SII lacking the first 130 amino acids was also cross-linked to the nascent RNA, but SII from Saccharomyces cerevisiae, which is inactive in concert with mammalian RNA polymerase II, failed to become photoaffinity labeled. SII-RNA contact was not detected after a labeled oligoribonucleotide was released from the complex by nascent RNA cleavage, demonstrating that this interaction takes place between elongation complex-associated but not free RNA. This shows that the 3'-end of RNA is near the SII binding site on RNA polymerase II and suggests that SII may activate the intrinsic RNA hydrolysis activity by positioning the transcript in the enzyme's active site.
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PMID:Elongation factor SII contacts the 3'-end of RNA in the RNA polymerase II elongation complex. 879 87

Fidelity of DNA and protein synthesis is regulated by a proofreading mechanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its control has been hampered by the necessity to employ complex DNA templates requiring either a promoter and initiation factors or 3'-extended templates. To circumvent this difficulty, we have created an RNA-DNA dumbbell template that can be recognized as a template-primer and extended by RNA polymerase II. By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out template-dependent elongation at the mispaired end. The transcripts containing misincorporated residues can be cleaved by the very slow 3'-->5' ribonuclease activity of the RNA polymerase II, but enhancement of this activity by the elongation factor TFIIS generates RNA with a high degree of fidelity. This enhanced preferential cleavage of misincorporated transcripts suggests an important role for TFIIS in maintaining transcriptional fidelity.
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PMID:Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. 894 93

RNA polymerase II contains a ribonuclease activity which is stimulated by the transcription elongation factor SII. This nuclease shortens the nascent RNA and facilitates relief of transcriptional arrest by allowing the enzyme to make multiple attempts to read through an obstacle to transcription. The catalytic center of this ribonuclease is unknown, although a region of the enzyme's second largest subunit shares local sequence similarly with barnase and other bacterial ribonucleases. To test the role of the barnase homology region in SII-activated cleavage, we engineered a single amino acid change in the Saccharomyces cerevisiae enzyme at a position homologous to a catalytic residue of barnase (Glu-371) and has been suggested as a participant in active site chemistry of RNA polymerase II. We purified RNA polymerase II from mutant yeast and assayed its ability to cleave and re-extend the nascent RNA following SII treatment. We find no defects in this function of the mutant enzyme, suggesting that the barnase homology region does not represent the active site of the SII-activated nuclease. These mutant yeast cells were also resistant to mycophenolic acid, which slows the growth of some yeast mutants bearing elongation defective RNA polymerase II or mutant elongation factor SII.
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PMID:Glutamic acid-371 of the barnase homology domain in RNA polymerase II is not required for SII-activated RNA cleavage. 903 12

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.
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PMID:Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1. 937 37


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