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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus. Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion. RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA. These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs. In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit. Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity. This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis. The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region. These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.
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PMID:A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes. 198 69

Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.
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PMID:Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex. 244 22

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.
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PMID:Mouse DNA polymerase alpha-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A1-2(C2-3/T2) sequences on a minute virus of mice DNA template. 385 59

We describe a new assay system that allows a rapid, direct, and quantitative detection of promoter-dependent in vitro transcription by RNA polymerase II. The template used is a hybrid plasmid containing the adenovirus major late promoter linked to a synthetic 400-base-pair DNA fragment that lacks cytidine residues on the transcribed strand--i.e., generates a transcript with no guanosine residues. In vitro transcriptions are carried out in the absence of GTP or, if the reactions contain GTP, in the presence of RNase T1 and the chain terminator 3'-0-methyl-GTP. Under these conditions the only RNAs that can accumulate, whether from a circular or linearized DNA template, are the 400-nucleotide RNase T1-resistant transcripts resulting from accurate initiation at the major late promoter. Thus, specific transcription can be directly monitored by conventional RNA quantitation methods. Using this fast assay, we show that three basic transcription factors, TFIIB, TFIID, and TFIIE, are absolutely required, in addition to the RNA polymerase II, for specific transcription initiation from the adenovirus major late promoter. Units of activity can be defined for each of these individual components. The applicability of this kind of assay to other systems is discussed.
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PMID:Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. 392 56

We have identified a single transcriptional initiation site for the glutamic tRNA and COB (cytochrome b) genes by using the complementary techniques of in vitro capping of RNA and in vitro transcription. In the capping reaction, mitochondrial RNA is labeled with [alpha-32P]GTP by vaccinia virus guanylyltransferase. This reaction is specific for the 5' ends of RNA retaining the terminal triphosphate of transcriptional initiation. Exploiting the extremely low G+C content (18%) of yeast mitochondrial DNA, we digested in vitro capped transcripts from various petite deletion mutants with the G-specific RNase T1. By petite deletion mapping, a capped transcript giving rise to a 51-base RNase T1-generated oligonucleotide was localized near the glutamic tRNA gene. When the sequence of this oligonucleotide was determined, it perfectly matched the DNA sequence 391 base upstream of the glutamic tRNA. Purified yeast mitochondrial RNA polymerase initiated transcription in vitro at the same site as shown by the sequence of the 33-base oligonucleotide product of the reaction performed in the absence of CTP. Initiation starts at a nonanucleotide sequence previously implicated in yeast mitochondrial transcriptional initiation. Because there is no evidence of an initiation site in the 1,050 bases between the glutamic tRNA and COB genes, the two genes are likely to be transcribed together. Further evidence of a long common transcript was provided by RNA blot hybridization.
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PMID:Identification of a single transcriptional initiation site for the glutamic tRNA and COB genes in yeast mitochondria. 613 68

The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed.
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PMID:The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study. 620 Apr 75

We have used vaccinia virus guanylyltransferase to label polyphosphate-terminated yeast mitochondrial RNAs in vitro with [alpha-32P]GTP. Hybridization of RNA labeled in vitro indicates the presence of multiple transcriptional initiation sites in both grande and petite mitochondrial genomes. Agarose/urea gel electrophoresis of capped RNA suggests the existence of a precursor to the small (14 S) rRNA. In contrast, direct examination of the large (21 S) rRNA by partial ribonuclease T1 digestion reveals a complete lack of processing of the 5' end of the primary transcript of this RNA.
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PMID:Transcriptional initiation and 5' termini of yeast mitochondrial RNA. 626 22

The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.
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PMID:The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr. 626 13

The 6 S leader RNA transcript from the Escherichia coli threonine operon controlling region was synthesized in vitro using purified RNA polymerase and restriction fragment DNA templates. The terminated leader transcript was analyzed by RNase T1 digestion followed by electrophoresis on 20% polyacrylamide, 8 M urea gels. Oligonucleotides of 7, 8, 13, 15, and 35 bases in length were detected and correlated with the known DNA sequence. The kinetics of RNase T1 digestion indicated that the RNA forms extensive secondary structure, especially at the 3'-terminus of the transcript. The sites of transcription initiation were determined by labeling the 5'-end of the transcript with [gamma-32P]ATP or -GTP followed by direct RNA sequencing. The DNA sequence preceding the initiation site shows homology with the equivalent regions of other bacterial and bacteriophage promoters. The transcription termination sites were determined by mapping of the RNase T1 oligonucleotides arising from the 3'-terminus of the transcript. Comparison of the mobilities of the 3'-oligonucleotides with the mobilities of standards on 20% polyacrylamide, 8 M urea gels indicated that the RNA contains a heterogeneous 3'-terminus. The two predominant oligonucleotides were CU7 and CU8. The 3'-terminus of the transcript also contains a region of dyad symmetry immediately preceding a stretch of uridine residues, characteristic of other rho-independent transcripts. In addition, kinetic studies indicated that RNA polymerase pauses approximately 50 base pairs upstream from the site of termination. The pause site appears to be immediately distal to another region of dyad symmetry.
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PMID:Initiation, pausing, and termination of transcription in the threonine operon regulatory region of Escherichia coli. 627 52

We have studied transcriptional initiation in the mitochondria of the yeast Saccharomyces cerevisiae by analyzing mitochondrial transcripts from grande and petite yeast after labeling in vitro with vaccinia virus guanylyltransferase and [alpha-32P]GTP. This procedure labels triphosphate-terminated RNA which arises from transcriptional initiation. Exploiting the extremely low GC content (18%) of yeast mitochondrial DNA, we digested the in vitro capped transcripts with the G-specific ribonuclease T1; this resulted in 27 oligonucleotides varying in size from 2 to 51 nucleotides. RNA from 14 overlapping petites was analyzed and 20 transcripts were localized by deletion mapping. Nineteen oligonucleotides were sequences and 13 were identified and precisely localized by comparison with known DNA sequences. In all cases, transcription is initiated at a consensus nonanucleotide sequence which can be considered part of the yeast mitochondrial promoter. We identified initiation sites for the 21 S and 14 S rRNAs; the phenylalanine, f-methionine, and glutamic tRNAs; two sites for the OLI-1 gene; and three for the ori (rep) regions. Most promoters appear to give rise to very long multigene primary transcripts. Examples are multigene transcripts for the glutamic tRNA and COB genes and for the OLI-1, serine tRNA, and Var genes. Since the consensus nonanucleotide sequences at the ori regions are similar to those at other transcriptional initiation sites, it is likely that the same RNA polymerase primes DNA replication and gene transcription.
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PMID:Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase. 631 17


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