Studies on transfer ribonucleic acids and related compounds. 22. Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61-77 from Escherichia coli tRNA <sup>fMet</sup>

Ohtsuka, Eiko; Nishikawa, Satoshi; Markham, Alexander F.; Tanaka, Shoji; Miyake, Toru; Wakabayashi, Toshiaki; Ikehara, Morio; Sugiura, Masahiro

doi:10.1021/bi00616a006

Cited by 28 publications

(11 citation statements)

References 24 publications

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“…Nearest-neighbor analysis (17), 3'-and 5'-terminal analysis (20), and partial nuclease P1 digestion (22) for mobility shift analysis were as described. For complete RNase T1 digestion ofthe product (1 pmol), RNase T1 (1 unit) was used in the presence of phosphatase (180 microunits) in (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The 3'-phosphorylated heptadecamer 4 was prepared as described (22). The 3'-phosphorylation of C-A-A-C-C-A (16) was done with P1-adenosine-P2-(o-nitrobenzyl) pyrophosphate and RNA ligase (19).…”

Section: Methodsmentioning

confidence: 99%

“…The 5'-terminal icosanucleotide (20), the tetradecanucleotide (bases 21-34) (21), and the 3'-heptadecanucleotide (22) have been obtained by this method. The 5'-quarter molecule here was found to reconstitute methionine.…”

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confidence: 99%

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Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

Ohtsuka¹,

Tanaka²,

Tanaka³

et al. 1981

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

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A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNA; except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths.of3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase andjoined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.Chemical synthesis ofnucleic acids has been a challenging problem in organic chemistry since the structure ofthe nucleic acids was elucidated. Chemical methods to synthesize short ribo-and deoxyribopolynucleotides with defined sequences were established in early 1960s, and those oligonucleotides were important in the elucidation of the genetic code (1). Discovery of DNA ligase allowed the synthesis of bihelical DNAs from chemically synthesized deoxyribopolynucleotides. With this chemical-enzymatic method the genes for yeast alanine tRNA (2) and Escherichia coli tyrosine tRNA precursor (3) have been synthesized; the latter was the first synthetic functional DNA molecule. Genes for peptides have also been synthesized by the same approach, and the methods for joining double-stranded DNA pieces with protruding ends have been used in various recently developed reactions for genetic manipulations.Although tRNAs are the smallest nucleic acids with unique functions, their synthesis has been difficult until recently, mainly because of the lack ofgood synthetic methods for larger .oligoribonucleotides as well as a lack ofjoining enzymes. After the primary structure of yeast alanine tRNA had been determined (4), the nona-and hexanucleotide corresponding to the terminal sequence of this tRNA were synthesized by phosphodiester block condensation. These fragments in turn were used to form reconstituted molecules with natural tRNA fragments derived by RNase digestions. However, aminoacylation was not possible because the synthetic fragments were too small to form sufficiently stable complexes for recognition by the alanyl-tRNA synthetase (5). The discovery of RNA ligase (6) and its ability to join single-stranded oligoribonucleotides (7) made it possible to elongate synthetic RNA fragments to yield larger molecules such as tRNAs.The initiator methionine tRNA of prokaryotes has a special role in protein biosynthesis, which. manifests itself in several unique properties of that tRNA (8). It was also the subject of detailed modif...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

Ohtsuka¹,

Tanaka²,

Tanaka³

et al. 1981

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

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“…Another possible reason for the difference between the proportion of 5Ј L vRNA deletions observed by sequencing and that observed by primer extension may be due to a nucleotide preference of the RNA ligase. Several studies have shown that the efficiency of the joining reaction is dependent on the particular donor (5Ј) and acceptor (3Ј) terminal nucleotides and the secondary structure of the termini (6,7,13,25,30). While purine acceptors and pyrimidine donors are generally believed to be the optimum substrates and could account for the large percentage of full-length L vRNAs ligated, not all types and combinations of oligoribonucleotides have been examined.…”

Section: Fig 3 Northern Hybridization Analysis Of Sr-11 Vrna Duringmentioning

confidence: 99%

Accumulation of Terminally Deleted RNAs May Play a Role in Seoul Virus Persistence

Meyer

Schmaljohn

2000

J Virol

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Two independent, long-term infections were analyzed to determine whether changes in viral replication could contribute to the establishment and/or maintenance of persistent Seoul virus infections. Infected cell cultures initially contained high levels of infectious virus and intracellular viral RNA that peaked between approximately 7 to 16 days postinfection and then gradually declined until day 26. After day 26, the viral titers and the levels of the small (S), medium (M), and large (L) viral RNAs varied cyclically until the end of the studies. The changes in the concentrations of the RNAs and titer were similar in pattern and appeared to result from changes in the regulation of replication. Neither internal deletions nor an accumulation of nucleotide changes were found in the RNAs. However, fine mapping and sequence analysis revealed short deletions in some of the RNAs in the conserved complementary terminal sequences believed to contain the signals for initiation of replication and transcription. Deletions at the 3 termini of S, M, and L virus-sense RNAs (vRNAs) accumulated during the acute phase of infection just before the time that the viral titer and the concentration of vRNAs and virus complementary-sense RNAs (cRNAs) began to decline. The absence of deletions at the 5 termini of the S, M, and L cRNAs suggests that the 3-deleted vRNAs may not be replication competent. Thus, as the percentage of 3-deleted vRNAs increase in the population, they could potentially compete with standard virus and downregulate viral replication. Deletions at the 3 L cRNA and 5 L vRNA termini were also observed, and the proportion of these deleted RNAs varied cyclically during the infections. We propose a model in which terminal nucleotide deletions arise by nuclease activity of the viral polymerase. In addition, we speculate that cleaved terminal fragments might be used as primers during replication, resulting in the repair of some of the deleted RNAs.The Hantavirus genus, in the family Bunyaviridae, is composed of a large group of enveloped negative-strand RNA viruses with a tripartite segmented genome (32). The three segments, small (S), medium (M), and large (L), encode the viral nucleocapsid (N), envelope glycoproteins (G1 and G2), and polymerase (L), respectively (33,35,36). Hantaviruses replicate in the cell cytoplasm, and replication is likely to proceed by mechanisms similar to those used by other negative-strand RNA viruses. That is, synthesis of mRNA and antigenome or virus complementary-sense RNA (cRNA) is initiated from the 3Ј terminus of viral RNA templates, and the synthesis of virus-sense RNA (vRNA) is initiated from the 3Ј terminus of cRNA templates. The signals for initiation of replication and transcription have not yet been defined but are believed to reside in the imperfect inverted repeat present at the termini of each genomic segment. This sequence is approximately 20 nucleotides long and is conserved among all members of the genus. Because bunyavirus ribonucleocapsids are circular (24, 26) and the termin...

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“…Polynucleotide kinase and E. coli alkaline phosphatase were obtained from Takara Shuzo and nuclease Sl from Sankyo. Other enzymes used for the analyses of products were obtained as in [14,19,20]. Crude E. coli aminoacyltRNA synthetase was purified as described [21].…”

Section: Ribooligonucleotidesmentioning

confidence: 99%

Modification of the amino acid acceptor stem of E. coli tRNA_f^Met by ligation of chemically synthesized ribooligonucleotides

et al. 1985

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The single-stranged region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNApa was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNApa, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine. Aminoacylation Formylation Elongated aminoacylation end Synthetic oligonucleotia'eTruncated aminoacylation end ligation

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Studies on transfer ribonucleic acids and related compounds. 22. Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61-77 from Escherichia coli tRNA ^fMet

Cited by 28 publications

References 24 publications

Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

Accumulation of Terminally Deleted RNAs May Play a Role in Seoul Virus Persistence

Modification of the amino acid acceptor stem of E. coli tRNA_f^Met by ligation of chemically synthesized ribooligonucleotides

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Studies on transfer ribonucleic acids and related compounds. 22. Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61-77 from Escherichia coli tRNA fMet

Cited by 28 publications

References 24 publications

Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

Accumulation of Terminally Deleted RNAs May Play a Role in Seoul Virus Persistence

Modification of the amino acid acceptor stem of E. coli tRNAfMet by ligation of chemically synthesized ribooligonucleotides

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Studies on transfer ribonucleic acids and related compounds. 22. Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61-77 from Escherichia coli tRNA ^fMet

Modification of the amino acid acceptor stem of E. coli tRNA_f^Met by ligation of chemically synthesized ribooligonucleotides