T4 RNA ligase: Substrate chain length requirements

Kaufmann, Gabriel; Klein, Theodore M.; Littauer, Uriel Z.

doi:10.1016/0014-5793(74)80385-6

Cited by 48 publications

(27 citation statements)

References 9 publications

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“…Interestingly, we observed that the T4 RNA ligase could ligate the nicked substrate with an efficiency comparable to that of the circularization reaction (Table II). This observation is in contrast with previous reports that showed a lack of ligation by this enzyme when the ends to be ligated were fully base paired (25).…”

Section: Resultscontrasting

confidence: 55%

The Mitochondrial RNA Ligase from Leishmania tarentolae Can Join RNA Molecules Bridged by a Complementary RNA

Blanc¹,

Alfonzo²,

Aphasizhev³

et al. 1999

Journal of Biological Chemistry

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A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K m of 25 ؎ 0.75 nM and a V max of 1.0 ؋ 10 -4 ؎ 2.4 ؋ 10 ؊4 nmol/ min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.RNA ligases are present in a large variety of organisms, but only a few have been implicated in specific metabolic pathways. For example, T4 RNA ligase repairs nicks in the anticodon domains of tRNAs in T4-infected Escherichia coli cells (1). In eukaryotes and Archea, a tRNA ligase is involved in tRNA splicing; this RNA ligase contributes to the maturation of tRNAs by joining tRNA half molecules generated by the removal of introns from tRNA precursors (2). Recently, a 2Ј-5Ј RNA ligase has been characterized in uninfected bacteria; the function of this RNA ligase remains unclear, but it may reveal an unexpected step in E. coli RNA metabolism (3). An RNA ligase has also been invoked for the final step of uridine insertion/deletion RNA editing in mitochondria of kinetoplastid protozoa (4).RNA editing in kinetoplastid mitochondria is a posttranscriptional maturation of pre-edited mRNA (5). This modification process consists of insertions and, to a lesser extent, deletions of U residues in the pre-edited mRNA, usually within coding regions. The information for the specific insertions or deletions is present as complementary sequences (allowing G-U base pairs) in short guide RNA molecules (gRNAs).1 The gRNAs are encoded by both the maxicircle and minicircle components of the mitochondrial DNA (kinetoplast DNA) of trypanosomatids (4). Evidence to date suggests that the modified enzyme cascade model (4 -9) is essentially correct, but many details remain to be established. A specific gRNA first forms a duplex region just downstream of the editing site. This is followed by a precise endonucleolytic cleavage at the first mismatched base (10, 11), addition of Us to the 3Ј end of the 5Ј cleavage fragment, trimming of non-base-paired Us by a 3Ј to 5Ј exonuclease, and finally a religation of the two cleavage fragments. An RNA ligase activity has been detected in isolated mitochondria of Leishmania tarentolae (12) and Trypanosoma brucei (13). Ligase activity sedimented as a major 20 S peak and a minor 10 S peak in glycerol gradients of mitochondrial extract from L. tarentolae. In T. brucei, mitochondrial ligase activity sedimented as two peaks of equal size (14). Two proteins of 50 and 45 kDa in both species were detected comigrating with the ligase activities. These two proteins could be adenylated by [␣-32 P...

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Section: Resultscontrasting

confidence: 55%

The Mitochondrial RNA Ligase from Leishmania tarentolae Can Join RNA Molecules Bridged by a Complementary RNA

Blanc¹,

Alfonzo²,

Aphasizhev³

et al. 1999

Journal of Biological Chemistry

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“…Experiments with synthetic oligoadenylates suggested that this enzyme would most efficiently circularize small RNAs in the range 10-30 nt (Kaufmann et al 1974). Thus, miRNAs with appropriate ends and lengths of 20-24 nt (Zamore and Haley 2005) make very good substrates for this reaction.…”

Section: Circularization Of Mirnas By T4 Rna Ligase and Circligasementioning

confidence: 99%

miR-ID: A novel, circularization-based platform for detection of microRNAs

Kumar¹,

Johnston²,

Kazakov³

2010

RNA

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MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 29-OMe groups at their 39-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 59-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 59-overlapping primers for detection of repetitive sequences by qPCR.

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“…These data may reflect a role of RNA ligase in posttranscriptional conversion of presumably host polyribonucleotides into novel tRNA species during T4 infection. Bacteriophage T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3], discovered by Silber et al (1), ligates in vitro 5'-P (donor) and 3'-OH (acceptor) termini of single-stranded polynucleotides in a reaction that utilizes ATP and requires magnesium (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). RNA ligase was recently identified by Snopek et al as the T4 gene 63 product (11), which had been previously implicated in T4 tail fiber attachment (12).…”

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confidence: 99%

“…4) Assuming that the products were tRNAs, we may tentatively conclude that at least some of them originated from preexisting host tRNA species. RNA ligase lacks base specificity towards either donor or acceptor termini (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). It seems likely, therefore, that the observed specific ligation pattern had been determined by specific formation of acceptor-donor pairs in the plasmolyzed infected cells.…”

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confidence: 99%

RNA ligase reaction products in plasmolyzed Escherichia coli cells infected by T4 bacteriophage.

David¹,

Vekstein²,

Kaufmann³

1979

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

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Searching for a physiological role of T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3] activity, we developed an acellular system of plasmolyzed Escherichia coli cells infected by T4 bacteriophage. Upon incubation of this system with 2p was transferred into a large number of polyribonucleotides, mostly up to 300-400 residues long. The bulk of 32P in the product polyribonucleotides was found in 5'-terminal phosphate groups, suggesting that they originated by a phosphorylation reaction catalyzed by the endogenous polynucleotide kinase (EC 2.7.1.78). Indeed, these products were not seen in an acellular system from uninfected cells, and their amount and complexity increased with the progress of infection. Analysis of the 32P-labeled olyribonucleotide products by gel electrophoresis, either before or after digestion with alkaline phosphatase (EC 3 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. § 1734 solely to indicate this fact.

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T4 RNA ligase: Substrate chain length requirements

Cited by 48 publications

References 9 publications

The Mitochondrial RNA Ligase from Leishmania tarentolae Can Join RNA Molecules Bridged by a Complementary RNA

The Mitochondrial RNA Ligase from Leishmania tarentolae Can Join RNA Molecules Bridged by a Complementary RNA

miR-ID: A novel, circularization-based platform for detection of microRNAs

RNA ligase reaction products in plasmolyzed Escherichia coli cells infected by T4 bacteriophage.

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