A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps

Cohen, Scott B.; Cech, Thomas R.

doi:10.1017/s1355838298001010

Cited by 10 publications

(8 citation statements)

References 14 publications

(14 reference statements)

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“…The dependence of ligation efficiency on the length of the gap separating the two bridged fragments is consistent with the results of Cohen and Cech (38), who quantitated by disulfide cross-linking the relative flexibility of an RNA bridged duplex consisting of three strands. They examined constructs in which a bridge RNA hybridized to two RNA molecules yielding a nick or single-stranded gaps of 1, 2, or 3 nt.…”

Section: Substratesupporting

confidence: 89%

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: Substratesupporting

confidence: 89%

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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“…The similar condensation of nicked-, gapped- and nicked-gapped-DNA is somewhat surprising, as the persistence length of nicked- and gapped-DNA duplexes have been reported to be significantly different (45–47,49,50). Given the observation that these three types of DNA condense similarly, one might conclude that reduced persistence length does not fully account for the difference in the condensation of nicked- and gapped-DNA with respect to continuous DNA duplex.…”

Section: Discussionmentioning

confidence: 98%

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

Sarkar

Conwell²,

Harvey³

et al. 2005

Nucleic Acids Research

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The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.

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“…62,106,107 If a conformational search were rate-limiting, lengthening the tether between the P1 duplex and the ribozyme in this manner is most simply expected to increase the degrees of freedom involved in the docking process and thus decrease the rate of docking. 108 However, k dock does not decrease due to this modification; instead, the rate of docking is increased by , tenfold (Table 1(A) and Figure 3(d)). This result provides experimental evidence against a rate-limiting conformational search for docking and supports instead the model of a kinetic trap slowing docking.…”

mentioning

confidence: 95%

Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a Large Structured RNA

Bartley

Zhuang

Das

et al. 2003

Journal of Molecular Biology

105

170

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A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps

Cited by 10 publications

References 14 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

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a Large Structured RNA

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