Synthesis and purification of single-stranded RNA for use in experiments with PKR and in cell-free translation systems

Pe'ery, T., Mathews, M. B. (April 1997) Synthesis and purification of single-stranded RNA for use in experiments with PKR and in cell-free translation systems. Methods, 11 (4). pp. 371-81. ISSN 1046-2023 (Print)

URL: http://www.ncbi.nlm.nih.gov/pubmed/9126552
DOI: 10.1006/meth.1996.0435

Abstract

The biosynthesis of RNA in vitro using bacteriophage RNA polymerases has opened up many avenues of research. Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays, PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation of protein synthesis. It can be activated by very low concentrations of dsRNA and inhibited by small structured RNAs or high concentrations of dsRNA. The best-studied inhibitor of PKR activation is adenovirus VA RNA1. Its gene was cloned into a plasmid under the control of the T7 RNA polymerase promoter, and the optimization of VA RNA transcription is described. A dsRNA by-product of the transcription reaction activates PKR in kinase autophosphorylation assays, and hence a purification protocol that allows the separation and removal of dsRNA contaminants was developed. A scheme to analyze the RNA product with specific nucleases is discussed. In a reticulocyte cell-free translation system the activation of PKR by dsRNA contaminating a synthetic mRNA preparation is likely to lead to shut-off of translation. An assay to directly visualize and measure the level of PKR phosphorylation in the lysate is detailed.

Item Type: Paper
Uncontrolled Keywords: Bacteriophage T7/genetics Cell-Free System Electrophoresis, Polyacrylamide Gel Protein Biosynthesis Protein-Serine-Threonine Kinases/ metabolism RNA, Messenger/genetics RNA, Viral/ chemical synthesis/isolation & purification Research Support, U.S. Gov't, P.H.S. eIF-2 Kinase
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > bacteriophage
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > bacteriophage
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > bacteriophage

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > dsRNA
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > phosphorylation
CSHL Authors:
Communities: CSHL labs
Depositing User: Kathleen Darby
Date: April 1997
Date Deposited: 08 May 2014 15:05
Last Modified: 08 May 2014 15:05
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29985

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