Localization of in vivo ribosome pause sites

Jeong Kook Kim, M. J. Hollingsworth

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A protocol for the localization of the 5' boundaries of in vivo ribosomal pausing sites has been developed. These mapping experiments combine two basic techniques. The first is the isolation of polysomal transcripts via centrifugation of tissue extracts through a sucrose cushion in the presence of translational elongation inhibitors. The second technique involves a micrococcal nuclease protection assay first developed by Wolin and Walter for in vitro-bound ribosomes (EMBO J. 7, 3559-3569, 1988). Using this method, the 5' boundaries of in vivo ribosomal pause sites were localized on spinach chloroplast mRNAs derived from the atpA gene. This method is easily adaptable to the identification of in vivo ribosomal pause sites from any organism. It could also be adapted to the localization of in vivo binding sites for other nucleic acid binding proteins.

Original languageEnglish
Pages (from-to)183-188
Number of pages6
JournalAnalytical Biochemistry
Volume206
Issue number1
DOIs
Publication statusPublished - 1992 Oct 22
Externally publishedYes

Fingerprint

Micrococcal Nuclease
Tissue Extracts
Centrifugation
Ribosomes
Nucleic Acids
Sucrose
Elongation
Assays
Carrier Proteins
Nuclease Protection Assays
Genes
Binding Sites
Messenger RNA
Spinacia oleracea
Chloroplasts
Experiments
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Localization of in vivo ribosome pause sites. / Kim, Jeong Kook; Hollingsworth, M. J.

In: Analytical Biochemistry, Vol. 206, No. 1, 22.10.1992, p. 183-188.

Research output: Contribution to journalArticle

Kim, Jeong Kook ; Hollingsworth, M. J. / Localization of in vivo ribosome pause sites. In: Analytical Biochemistry. 1992 ; Vol. 206, No. 1. pp. 183-188.
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