Abstract
Abstract Incubation of pure yeast phenylalanine transfer RNA with high levels of ribonuclease T1 at 37° in 20 mm MgCl2 leads initially to the production of a 3' three-quarter-molecule and a 5' quarter-molecule as a result of rapid cleavage in the dihydrouridine loop. Subsequently, scission in the ribothymidine loop occurs, generating 3' quarter-molecules and half-molecules; the latter comprise the central half of the molecule extending from the dihydrouridine loop to the ribothymidine loop. The identity of these fragments was established by column fingerprinting of the oligonucleotides produced by complete digestion with ribonuclease T1. The central half-molecule, the 5' quarter-molecule, and the 3' quarter-molecule can be reannealed to form an aggregate the size of intact tRNA as judged by Sephadex G-100 chromatography. This reconstituted tRNAphe can be charged to approximately 20% with phenylalanine by partially purified phenylalanyl-tRNA synthetase despite the cleavages in the dihydrouridine and ribothymidine loops, thus indicating that these loops need not be intact for enzyme recognition. Neither individual fragments nor aggregates with large sections of the tRNA missing could be aminoacylated.
Highlights
Incubation of pure yeast phenylalanine transfer RNA with high levels of ribonuclease Tr at 37” in 20 mM MgClz leads initially to the production of a 3’ three-quarter-molecule and a 5’ quarter-molecule as a result of rapid cleavage in the dihydrouridine loop
We have studied the degradation of yeast tRNA Phe by ribonuclease T1 and demonstrated cleavage into large fragments the size of half- and quarter-molecules; these fragments recombine upon mixing to produce an aggregate that accepts phenylalanine and is the size of intact tRNA [12]
This material was preparatively aminoacylated with 14C-phenylalanine and rechromatographed on benzoylated DEAE-cellulose according to the method of Litt [14], to yield a symmetrical peak of pure tRNAPhe
Summary
Incubation of pure yeast phenylalanine transfer RNA with high levels of ribonuclease Tr at 37” in 20 mM MgClz leads initially to the production of a 3’ three-quarter-molecule and a 5’ quarter-molecule as a result of rapid cleavage in the dihydrouridine loop. Scission in the ribothymidine loop occurs, generating 3’ quarter-molecules and half-molecules; the latter comprise the central half of the molecule extending from the dihydrouridine loop to the ribothymidine loop. The identity of these fragments was established by column “fingerprinting” of the oligonucleotides produced by complete digestion with ribonuclease TX
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