Specificity of RNase U2*

Uchida, Tsuneko; Arima, Terukatsu; Egami, Fujio

doi:10.1093/oxfordjournals.jbchem.a129239

Cited by 75 publications

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“…(c) The T-Y bond is not cleaved by RNase Phyl in contrast to U-U bonds (e.g. U12-U13); this resistance may in part be due to the presence of Y adjacent to T. (d) The m'A-A bond is not cleaved by RNaseUz as expected [38].…”

Section: Oligonucleotides Present In Complete Endonuelease Digests Ofmentioning

confidence: 90%

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Randerath

Gupta

Chia

et al. 1979

European Journal of Biochemistry

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A second major species of leucine tRNA, t R N A e G (formerly designated t R N A e A ) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase TI digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNAscAA (tRNAP").The primary structure of baker's yeast tRNA?lG was elucidated by application of sensitive radioactive isotope derivative ('postlabeling') methods. Complete RNaseTl and A and partial RNase Uz fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2I-O-Methylated guanosine was characterized using the [y-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the RNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis.A different sequencing approach, consisting of a combination of 'readout sequencing' with tritium sequencing of complete RNase TI and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNases TI, UZ, and Phy, and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 unit of RNA and the analysis of the complete fragments about 6 A260 units.Interesting structural features of tRNAfLG are (a) the location of unique, leucine tRNA isoacceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA3cA,, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

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Section: Oligonucleotides Present In Complete Endonuelease Digests Ofmentioning

confidence: 90%

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Randerath

Gupta

Chia

et al. 1979

European Journal of Biochemistry

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“…RNA samples eluted from polyethylenimine-cellulose plates were further digested with pancreatic RNase (cleaves after Cp or Up), RNase T2 (cleaves after each Np), nuclease P1 [cleaves between the ribose and the phosphate at the 3' side and produces pN nucleotides (5'-phosphonucleosides)], or nuclease U2 (cleaves after Ap or Gp). The products of those digestions were analyzed as described (22)(23)(24).…”

Section: Methodsmentioning

confidence: 99%

Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

Gurevitz¹,

Jain²,

Apirion³

1983

Proc. Natl. Acad. Sci. U.S.A.

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A precursor molecule for lOSb (MI) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the lOSb RNA gene. The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E-mutant at the nonpermissive temperature. The RNA precursor includes lOSb RNA and an extra 3' fragment that contains a termination stem and loop. It can be processed in vitro to a molecule the size of lOSb RNA. None of the four endoribonucleases of E. coli-RNase III, RNase E, RNase F, or RNase P-takes part in this cleavage reaction. Therefore, we suggest that the processing of the precursor-IOSb RNA to iOSb RNA is carried out by a thus-far unidentified endoribonuclease. The accumulation of a RNA molecule in a RNase E-mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E. coli cell.Processing of RNA is an important aspect of RNA metabolism that plays a key role in the determination of the final population of RNA molecules in the cell. RNA processing involves a set of reactions that happen post-transcriptionally and prepares a RNA molecule for its function. The involvement of the three endonucleolytic RNA processing enzymes RNase III, RNase E, and RNase P has been well documented in the processing of Escherichia coli rRNA and tRNA (1-6).The processing enzyme RNase P is unique among all RNA processing enzymes because it consists of a protein and a RNA moiety (7-12). The RNA, MI, is required for RNase P activity (9) and is identical to the 10Sb RNA that accumulates in a strain containing the plasmid pL2 (pLN2) that partially complements the rnpA49 mutation (13,14). Introduction of the pL2 plasmid to E. coli strains leads to the accumulation of 1OSb (Ml) RNA, which is not a primary transcript (12)(13)(14).In the studies reported here we show that 10Sb (Ml) RNA, the RNA of RNase P, is derived from a precursor molecule (plOSb) that contains extra sequences at the 3' end, and we have established conditions for the in vitro processing of this precursor RNA. These experiments helped to identify a previously unrecognized RNA processing activity in E. coli. This precursor accumulates in a strain defective in the RNA processing enzyme RNase E, even though the enzyme RNase E does not process this RNA molecule. Therefore, the finding that this precursor accumulates in a RNase E-strain strengthens the concept of a complex of RNA processing enzymes in the E. coli cell.MATERIALS AND METHODS Strains. The rnp+ strain used was PP113 (15) and the rnpA49 strain was N2020 (16). The rne strain was N3441, which was derived from strain N3431 (17). To these strains the plasmid pL2 was introduced. This plasmid contains E. coli DNA that carries the gene for lOSb RNA (13). N2069 is a RNase III-strain derived from strain N7060 (18).Growth and Labeling of Cells. Cells were grown in lowph...

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“…The mechanism consists of two reactions, the transfer of the phosphate group to the 2'-hydroxyl group of the purine nucleotides and the hydrolysis to produce 3'-phosphate of the purine nucleoside (Arima et al, 1968;Uchida et al, 1970). Earlier studies had shown that RNAase U2 was a single-chain polypeptide consisting of 115 amino acid residues devoid of lysine and methionine and that it had half-cystine at the Nterminus and serine at the C-terminus (Uchida & Sato, 1973a).…”

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confidence: 99%

The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena

Sato

Uchida

1975

Biochemical Journal

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1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined. 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes;

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Specificity of RNase U2*

Cited by 75 publications

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Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena

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Specificity of RNase U2*

Cited by 75 publications

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Yeast tRNALeuUAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Yeast tRNALeuUAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena

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Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods