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)

A nickel complex has been shown to promote conformation-specific oxidation of guanosine in polynucleotide RNA. In all cases, reaction was strictly dependent on the solvent exposure and surface properties of guanine N7. Modification of native tRNA(Phe) (yeast) was detected at G18, G19, G20, and Gm34 and concurred with predictions based on its crystal structure. Additional guanine derivatives became exposed to oxidation only after the tRNA unfolded in the absence of Mg2+. Reaction of the Tetrahymena group I intron RNA (L-21 ScaI) also compared favorably to its three-dimensional model by appropriately identifying guanosine residues in hairpin loops, duplex termini, and the essential cofactor binding site. These results complemented prior data generated by hydroxyl radical, and in combination they served to distinguish the solvent accessibility of sugar backbone and base positions in guanosine residues. Most importantly, this nickel complex exhibited greater selectivity than either dimethyl sulfate or RNase T1 for characterizing tRNA(Phe) and intron RNA.
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PMID:A highly sensitive probe for guanine N7 in folded structures of RNA: application to tRNA(Phe) and Tetrahymena group I intron. 834 71

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.
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PMID:Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. 849 49

Initiation of reverse transcription is a crucial step of retroviral infection. In HIV-1, it involves hybridization of the 18 3'-terminal nucleotides of the primer tRNA3(Lys) to the primer binding site (PBS) of the viral RNA. Moreover, additional interactions between the two RNAs were recently evidenced [Isel et al. (1995) J. Mol. Biol. 247, 25269-25272]. To get further information on the topology of the viral RNA/tRNA3(Lys) complex, we used psoralen to induce RNA-RNA crosslinking. A defined intermolecular crosslinked complex was obtained. The crosslinked regions were characterized by RNase T1 digestion followed by bi-dimensional gel electrophoresis. The crosslinked residues (nucleotide mcm5S2U34 and U35 in the anticodon loop of tRNA3(Lys) and UCU154 in the viral RNA upstream of the PBS) were mapped using a retardation method coupled with random hydrolysis. The formation of this crosslink depends on the same elements that are required for the formation of the extended interactions between primer and template RNAs, i.e., the modified bases of the tRNA and a conserved A-rich loop located upstream of the PBS in the genomic RNA. Therefore, the present crosslinking data provide relevant information on the topology of the template/primer binary complex.
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PMID:Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys). 860 65

The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli. In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of [32P]AMP attached to MTF. Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II*. These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy. Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link. Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link. The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base.
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PMID:Lysine 207 as the site of cross-linking between the 3'-end of Escherichia coli initiator tRNA and methionyl-tRNA formyltransferase. 903 Jun 4

Drosophila RNase P and 3'-tRNase endonucleolytically process the 5' and 3' ends of tRNA precursors. We examined the processing kinetics of normal substrates and the inhibitory effect of the tRNA product on both processing reactions. The product is not a good RNase P inhibitor, with a KI approximately 7 times greater than the substrate KM of approximately 200 nM and is a better inhibitor of 3'-tRNase, with a KI approximately two times the KM of approximately 80 nM. We generated matrices of substitutions at positions G18/U55 and G19/C56 (two contiguous universally conserved D/T loop base pairs) in Drosophila tRNAHis precursors. More than half the variants display a significant reduction in their ability to be processed by RNase P and 3'-tRNase. Minimal substrates with deleted D and anticodon stems could be processed by RNase P and 3'-tRNase much like full-length substrates, indicating that D/T loop contacts and D arm/enzyme contacts are not required by either enzyme. Selected tRNAs that were poor substrates for one or both enzymes were further analyzed using Michaelis-Menten kinetics and by structure probing. Processing reductions arise principally due to an increase in KM with relatively little change in Vmax, consistent with the remote location of the sequence and structure changes from the processing site for both enzymes. Local changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.
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PMID:Matrices of paired substitutions show the effects of tRNA D/T loop sequence on Drosophila RNase P and 3'-tRNase processing. 942 63

Liquid chromatography/electrospray mass spectrometry is one of the rapidly developing techniques with which mass of large hydrophilic polymers such as proteins and nucleic acids can be determined precisely. The technique was applied to studies on the modifications of tRNAs. Various tRNA species purified from Escherichia coli were directly injected into a capillary reversed-phase column and the desalted and concentrated tRNAs were analyzed on-line with an electrospray mass spectrometer. In some cases, small but significant differences were noted between the theoretical and observed molecular masses, suggesting that there exist still unknown modifications. Under high resolution measurements, multiple peaks corresponding to species modified to a varying extent were resolved. To study the structures in detail, the isolated tRNA species were digested with ribonuclease T1, and the resulting mixture of fragments were analyzed by the same liquid chromatography/mass spectrometry. In this way, most of the fragments were easily identified solely from their masses, and the positions where the expected and real structures differ were revealed. The results obtained showed the presence of micro-heterogeneity among tRNAs and demonstrated at the same time the power of the hyphenated technique for the structural analysis on nucleic acids.
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PMID:A liquid chromatography/electrospray mass spectrometric study on the post-transcriptional modification of tRNA. 949 Jul 95

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.
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PMID:Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches. 960

Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification. Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are required for subunit assembly. Experimental difficulties in the mapping of ribose methylations increase with RNA molecular size and the complexity of mixtures resulting from nuclease digestion. A new and relatively rapid approach based on tandem mass spectrometry is described in which any of four ion reaction pathways occurring in the mass spectrometer can be monitored which are highly specific for the presence of 2'-O -methylribose residues. These pathways emanate from further dissociation of ribose-methylated mononucleotide (Nmp) ions formed in the electrospray ionization region of the mass spectrometer to then form the base, methylribose phosphate or PO(3)(-)anions. The mass spectrometer can be set for detection of generic ribose methylation (Nm) in oligonucleotides, selectively for each of the common methylated nucleo-sides Cm, Gm, Am or Um or for specific cases in which the base or sugar is further modified. By direct combination of mass spectrometry with liquid chromatography the method can be applied to analysis of complex mixtures of oligonucleotides, as for instance from synthetic or in vitro reaction mixtures or from nuclease digests of RNA. An example is given in which the single ribose-methylated nucleoside in Escherichia coli 16S rRNA (1542 nt), N(4),O-2'-dimethylcytidine, is detected in 25 pmol of a RNase T1 digest and localized to the fragment 1402-CCCGp-1405 in a single 45 min analysis.
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PMID:Selective detection of ribose-methylated nucleotides in RNA by a mass spectrometry-based method. 1047 50

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.
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PMID:A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors. 1184 97

Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNA(TyrII) from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications.
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PMID:Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry. 1246 67


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