High-Speed Preparative-Scale Separation and Purification of Ribosomal 5S and 5.8S RNA'S via Sephacryl S-300 Gel Filtration Chromatography

Abstract

In this work we present a rapid and economical alternative to Sephadex and high performance size exclusion chromatography (HPSEC) for preparative-scale separation and purification of low molecular weight RNA's: 5.8S RNA, 5S RNA, and tRNA's. These three RNA species can be well resolved from each other and from higher molecular weight RNA species via Sephacryl S-300 gel filtration chromatography under mild eluting conditions: 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl. For a sample load of about 250 mg, the resolving power of a Sephacryl S-300 column (78 X 3.2 cm) is comparable to that of a 4.5 times larger Sephadex G-75 column (144 X 5 cm). Moreover, the total separation period is 2.5 times shorter for the Sephacryl method. Up to 500 mg or more of crude ribosomal RNA mixtures could be separated via two Sephacryl S-300 columns operated in tandem.

Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

“…Further the RNA concentration was evaluated using Nanodrop spectrophotometer. The bulk tRNA was purified as described [57]. Heparin was purchased from Lovens Kemiske Fabrik (Denmark) and BSA from Promega.…”

Over Expression of a tRNALeu Isoacceptor Changes Charging Pattern of Leucine tRNAs and Reveals New Codon Reading

“…32 [ 3 H]fMet-tRNA fMet was prepared from tRNA bulk as described. 31 tRNA Leu isoacceptors were purified from tRNA bulk by BD-Sepharose chromatography with minor modifications as described.…”