[9] Site-specific cleavage of transcript RNA

Lapham, Jon; Crothers, Donald M.

doi:10.1016/s0076-6879(00)17011-9

Cited by 18 publications

(15 citation statements)

References 18 publications

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“…This yields the site-specific cleavage of the RNA in the complex at the 5Ј-end of the chimera (28,29). We believe that the faint bands that were occasionally seen above the main products (see, for example, lanes 2, 6, and 10 of Fig.…”

Section: Methodsmentioning

confidence: 99%

RNA Polymerase II Transcription Complexes May Become Arrested If the Nascent RNA Is Shortened to Less than 50 Nucleotides

Újvári

Pal

Luse

2002

Journal of Biological Chemistry

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A significant fraction of RNA polymerase II transcription complexes become arrested when halted within a particular initially transcribed region after the synthesis of 23-32-nucleotide RNAs. If polymerases are halted within the same sequence at a promoter-distal location, they remain elongation-competent. However, when the RNAs within these promoter-distal complexes are truncated to between 21 and 48 nucleotides, many of the polymerases become arrested. The degree of the arrest correlates very well with the length of the RNA in both the promoter-proximal and -distal complexes. This effect is also observed when comparing promoter-proximal and promoter-distal complexes halted over a completely different sequence. The unusual propensity of many promoter-proximal RNA polymerase II complexes to arrest may therefore be recreated in promoter-distal complexes simply by shortening the nascent RNA. Thus, the transition to full elongation competence by RNA polymerase II is dependent on the synthesis of about 50 nt of RNA, and this transition is reversible. We also found that arrest is facilitated in promoter-distal complexes by the hybridization of oligonucleotides to the transcript between 30 and 45 bases upstream of the 3-end.There is increasing evidence for eukaryotic gene regulation during the process of transcript elongation (reviewed in Ref. 1). It is thus important to understand in detail the elements that affect the efficiency of elongation by RNA polymerase II transcription complexes. Both biochemical (reviewed in Ref.2) and structural studies of prokaryotic and eukaryotic RNA polymerases support a particular model of the transcript elongation complex (3-7). According to this model, ternary complexes are stabilized primarily by the presence of an 8 -9-bp RNA-DNA hybrid just upstream of the catalytic center of the RNA polymerase and by a domain of the polymerase that serves as a sliding clamp, encircling the DNA just downstream of the active site and preventing dissociation of the template from the complex.This model explains certain examples of sequence-specific loss of elongation competence, or arrest, by multisubunit RNA polymerases. For example, RNA polymerase II arrests primarily at the boldfaced Ts while traversing the T Ia sequence (nontemplate strand) TTTTTTTCCCTTTTTT from the histone H3.3 gene (8). Complexes with U-rich 3Ј-ends contain the weakest possible RNA-DNA hybrid (U:dA). In response to this situation, the polymerase can translocate upstream, carrying the transcription bubble to a location where a more stable RNA-DNA hybrid forms. Indeed, an upstream translocated footprint was reported for polymerases arrested at the histone H3.3 T Ia site (9, 10). Upstream translocation removes the 3Ј-end of the RNA from the active site and thus prevents transcription from continuing. Arrested complexes can be restarted by cleavage of the transcript such that the 3Ј RNA end is again aligned with the enzyme catalytic center. This cleavage is catalyzed by the active site of the polymerase and is highly stimulated by ...

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Section: Methodsmentioning

confidence: 99%

RNA Polymerase II Transcription Complexes May Become Arrested If the Nascent RNA Is Shortened to Less than 50 Nucleotides

Újvári

Pal

Luse

2002

Journal of Biological Chemistry

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“…Cleavage results in a 5'-hydroxyl group and a 2',3'-cyclic phosphate similarly to small ribozymes. Moreover, it has been shown that RNAs can be cleaved sequence-specifically by RNase H, when the RNA of interest is hybridized with a 2'-O-methyl-RNA/DNA chimera [118,119]. In contrast to ribozyme and DNAzyme mediated RNA cleavage, RNase H produces 5'-monophosphates and 3'-hydroxyl groups.…”

Section: Rna Synthesismentioning

confidence: 99%

“…The tRNA scaffold can be removed either by DNAzymes or by sequence-specific RNase H cleavage [115,[117][118][119]. Using this method, a reasonable amount of RNA for NMR studies (0.8 µmoles)…”

Section: Rna Synthesismentioning

confidence: 99%

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

Dominguez

Schubert

Duss

et al. 2011

Progress in Nuclear Magnetic Resonance Spectroscopy

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“…Although intriguing, these bands are generally so faint that they are neglected. Recently, we found that this phenomenon was more prominent than usual when purifying by denaturing PAGE a 14-mer chimeric 2Ј-O-methyl/DNA oligonucleotide intended for use in site-directed RNA cleavage with Escherichia coli RNase H (Lapham and Crothers, 2000). Therefore, we decided to take this opportunity to elucidate the structure of this by-product and its mechanism of formation.…”

Section: Introduction Smentioning

confidence: 99%

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

2006

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During the synthesis of oligonucleotides by the standard phosphoramidite method using 2-deoxycytidine-derivatized solid support, a side reaction was observed that gave rise to the formation of high molecular weight N-branched oligomers having two identical chains linked to the 3-terminal 2-deoxycytidine. Postsynthesis treatment with neat triethylamine trihydrofluoride selectively cleaved the phosphoramidate linkage and converted the N-branched oligomers back to the expected oligonucleotides.

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[9] Site-specific cleavage of transcript RNA

Cited by 18 publications

References 18 publications

RNA Polymerase II Transcription Complexes May Become Arrested If the Nascent RNA Is Shortened to Less than 50 Nucleotides

RNA Polymerase II Transcription Complexes May Become Arrested If the Nascent RNA Is Shortened to Less than 50 Nucleotides

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

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