Abstract
Abstract A new type of fractionating step involving gel filtration is introduced for the purification of certain transfer ribonucleic acids (tRNAs). It is based on the increase that occurs in the molecular dimensions of these tRNAs when they are trapped, reversibly, in a denatured state under conditions where the other tRNA components of the mixture are in the native conformation. This step, coupled to countercurrent distribution, is used to obtain in a near homogeneous state a leucine-specific tRNA that cannot be so purified by countercurrent distribution alone, as several other tRNA species have very similar partition coefficients in the two-phase solvent system employed. The applicability of the same techniques for the partial purification of a methionine-, an arginine-, and a glutamine-specific tRNA from yeast is also demonstrated. The potential of this combination of methods for large scale fractionation of tRNA is discussed.
Highlights
The transfer ribonucleic acid fraction from a cell extract consists of a mixture of more than 40 tRNA1 species
While most of them have specific biological roles not shared by other transfer ribonucleic acids (tRNAs), they are all very similar in charge, molecular weight [1, 2], and in the conformation of their native states [3, 4]
When enzymatic replacement of the terminal AMP residue was performed after the first countercurrent distribution, it did not appreciably alter the profile of leucine-acceptor activity of the redistribution; the three major tRNALeU peaks were still clearly separated
Summary
TRN.4-Large quantities of unfractionated yeast tRNA (bakers’ yeast, Standard Brands) were prepared according to the method of Holley [20].CountercurrentDistribution~TwoCraig-Post type machines were employed, one with 60 tubes, lOO/lOO ml, for preliminary bulk separations, and one with 340 tubes, lo/10 ml, for finer separations of partially fractionated samples. TRN.4-Large quantities of unfractionated yeast tRNA (bakers’ yeast, Standard Brands) were prepared according to the method of Holley [20]. Distributions were performed at the indicated temperatures kO.5” with either of two different phosphate buffer-formamide-isopropanol solvent systems, here designated Solvent I [21] and Solvent II [22], to which EDTA was added to 0.001 M [16]. Gel ZXtratiolz--Dry Sephadex G-100 (Pharmacia Fine Chemicals, Inc.) was added slowly, under vigorous stirring, to a large volume of 0.05 M potassium phosphate, pH 6.8, and the mixture was boiled for 3 hours. The gel was allowed to settle for 10 min. The gel was washed five times with hot distilled water, and equilibrated with solvent
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