[14] Preparation of specific ribosomal RNA fragments

Draper, David E.; White, Su; Kean, Joanne M.

doi:10.1016/s0076-6879(88)64045-6

Cited by 48 publications

(36 citation statements)

References 14 publications

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“…RNA Transcription-Linear plasmid DNA was transcribed using procedures outlined by Gurevich (13) or described previously (14). Radiolabeled RNA used in nitrocellulose filter binding assays was purified using Nensorb reverse phase columns (14).…”

Section: Methodsmentioning

confidence: 99%

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Translational Repression of the Escherichia coli α Operon mRNA

Schlax¹,

Xavier²,

Gluick³

et al. 2001

Journal of Biological Chemistry

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Ribosomal protein S4 represses synthesis of the four ribosomal proteins (including itself) in the Escherichia coli ␣ operon by binding to a nested pseudoknot structure that spans the ribosome binding site. A model for the repression mechanism previously proposed two unusual features: (i) the mRNA switches between conformations that are "active" or "inactive" in translation, with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA ("entrapment"), in contrast to direct competition between repressor and ribosome binding ("displacement"). These two key points have been experimentally tested. First, it is found that the mRNA pseudoknot exists in an equilibrium between two conformers with different electrophoretic mobilities. S4 selectively binds to one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms. Second, we have used S4 labeled at a unique cysteine with either of two fluorophores to characterize its interactions with mRNA and 30 S subunits. Equilibrium experiments detect the formation of a specific ternary complex of S4, mRNA pseudoknot, and 30 S subunits. The existence of this ternary complex is unambiguous evidence for translational repression of the ␣ operon by an entrapment mechanism.

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Section: Methodsmentioning

confidence: 99%

“…Radiolabeled RNA used in nitrocellulose filter binding assays was purified using Nensorb reverse phase columns (14). RNA used in gel mobility experiments was excised from denaturing polyacrylamide gels and extracted by either electroelution (Schleicher & Schuell Elutrap) or soaking crushed gel in buffer.…”

Section: Methodsmentioning

confidence: 99%

Translational Repression of the Escherichia coli α Operon mRNA

Schlax¹,

Xavier²,

Gluick³

et al. 2001

Journal of Biological Chemistry

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“…Separation of desired RNA product from cis-acting ribozyme T7 RNA polymerase tends to add extra nucleotides to the 39 end of synthesized RNA oligonucleotides (Milligan et al 1987;Draper et al 1988;Pleiss et al 1998), often yielding RNA oligonucleotides with heterogeneous ends. As a single RNA species is typically required for high-resolution structural studies of RNAs or RNA d protein complexes, this behavior is problematic.…”

Section: Fplc Size-exclusion Column Selection (Superdex 200 or Superdmentioning

confidence: 99%

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

Kim

McKenna²,

Puglisi³

et al. 2006

RNA

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We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.

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“…Although T7 RNA polymerase tends to add extra nucleotides to the 3Ј-end of the desired RNA (Milligan et al 1987;Draper et al 1988;Pleiss et al 1998), this problem has been largely overcome through the use of cis-acting ribozymes at the 5Ј-and 3Ј-ends of the RNA of interest (Price et al 1995;Ferre-D'Amare and Doudna 1996) or through the use of synthesized, partially 2Ј-O-methyl-modified DNA templates (Kao et al 1999). The transcription product RNAs are purified by preparative denaturing polyacrylamide gel electrophoresis, eluted from the gel matrix, concentrated, and refolded.…”

Section: Introductionmentioning

confidence: 99%

A general method for rapid and nondenaturing purification of RNAs

Kieft¹,

Batey²

2004

RNA

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A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant H␦V ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.

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[14] Preparation of specific ribosomal RNA fragments

Cited by 48 publications

References 14 publications

Translational Repression of the Escherichia coli α Operon mRNA

Translational Repression of the Escherichia coli α Operon mRNA

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

A general method for rapid and nondenaturing purification of RNAs

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