Specific Degradation of a Plant Leucyl Transfer Ribonucleic Acid by a Factor in the Homologous Synthetase Preparation

tRNAI-U in Pisum sativum seed has been purified. This tRNA species contains a cytokinin-active nucleoside and accounts for approximately 7% of the total cytokinin activity in acid hydrolysates of pea tRNA. Tle cytokinin has been identified as ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-f3-D-ribofuranosylpurine.The modified nucleoside c-io6A3 was first tentatively identified by Hall et al. (7) in tRNA preparations from peas, corn, and spinach and has since been isolated from tRNAs of several other plants (6,12). It has also been reported, on the basis of bioassays, to occur in tRNA preparations of certain bacterial species;i.e. Corynebacterium fascians (8), a plant pathogen, and in species of Rhizobium (10), the bacterium associated with nitrogen-fixing nodules in legumes. The 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-,8-D-ribofuranosylpurine, t-io6A, the isomer generally found in free form in plants, has also been isolated from Pisum tRNA (14).Gas chromatographic evidence for the presence of c-io6A in tRNA'U from peas has been reported (2). This paper reports the isolation of this tRNA species and the definitive identification of c-io6A as its cytokinin-active nucleoside constituent.MATERIALS AND METHODS Purification of Leucyl tRNA. Pea seeds (Pisum sativum cv. Alaska) that had been surface-sterilized and soaked for 48 hr in aerated, sterile distilled H20 were homogenized with 1 ml/g fresh weight of 0.1 M tris-HCl (pH 7.8), 0.1 mm EDTA, 0.2% SDS, and then this homogenate was extracted twice with equal volumes of phenol saturated with the same buffer. The final aqueous phase was adjusted to 0.1 M NaCl and the RNA was precipitated by addition of 2 volumes of cold 95% (v/v) ethanol. The RNA was then suspended in 0.02 M sodium acetate (pH 4.5), 1 mm EDTA, and applied to a column of DEAE-cellulose previously equilibrated with the same buffer. After washing the column with 5 bed volumes of buffer containing 0.3 M NaCl, the tRNA was eluted with 2.5 bed volumes of the same buffer containing 1 M NaCl.The tRNA was applied to a column of BD-cellulose (5) Bioassay of Cytokinin Activity. The fractionation of plant tRNA species containing c-io6A was monitored with the tobacco callus bioassay, as this compound is active as a cytokinin in the free form (11). The tRNA fractions were dissolved in 10 ml of distilled H20, acid-hydrolyzed, and incorporated in serial dilutions into nutrient medium (12). Cytokinin content is expressed either as kinetin equivalents (KE, where 1 KE is the growth response obtained on medium containing 1 ,ug/1 kinetin) or as ,umol of c-io6A calculated by comparison of the growth response with the response given by acid-hydrolyzed samples of this nucleoside tested in serial dilutions.Determination of Leucine Acceptor Activity. The leucyl tRNA synthetase was prepared from soaked pea seeds by the procedure of Scott and Morris (10) except that the chromatography step on Bio-Gel P-150 was omitted. Reaction mixtures for aminoacylation were incubated at 30 C and contained tR...

mentioning

confidence: 77%

mentioning

confidence: 87%

Ribosyl-cis-zeatin in a Leucyl Transfer RNA Species from Peas

Einset

Swaminathan²,

Skoog³

1976

“…The most detailed investigations of the distribution of cytokinin moieties with respect to individual tRNA species have been conducted with tRNA from microbial sources, but all available evidence concerning cytokinin distribution in tRNA from higher plant and animal tissues is consistent with this concept (2,6,12,(19)(20)(21). Not all tRNA species within the U group necessarily contain a cytokinin moiety.…”

Section: Methodsmentioning

confidence: 94%

“…tRNALu species from peas (2,6) and soybeans (12), but the other U-group tRNA species have not been examined in these plant materials. In Euglena (4,11) and Phaseolus vulgaris (8,9), the cytokinin 2-methylthio-N6-(A2-isopentenyl)adenosine has been identified as a constituent of the corresponding chloroplast tRNAPhe species.…”

mentioning

confidence: 99%

Cytokinin-Active Ribonucleosides in Phaseolus RNA

Edwards

Armstrong²

1981

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-ceilulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNACYs, tRNATr, tRNATyr, a major peak of tRNAPh, and a large fraction of tRNALU were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNASr species and with minor tRNALU species. In adtion, several Anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected. tRNALu species from peas (2, 6) and soybeans (12), but the other U-group tRNA species have not been examined in these plant materials. In Euglena (4, 11) and Phaseolus vulgaris (8, 9), the cytokinin 2-methylthio-N6-(A2-isopentenyl)adenosine has been identified as a constituent of the corresponding chloroplast tRNAPhe species.We report here the results of an examination of the distribution of cytokinins in tRNA species from etiolated Phaseolus vulgaris seedlings. Etiolated material was selected for this study to minimize the contribution of plastid tRNA species to the distribution of cytokinin activity and to provide a more direct comparison with the results obtained earlier for wheat germ tRNA. As in the case of wheat germ tRNA (20), the distribution of cytokinin moieties in tRNA species from etiolated bean seedlings appears to be more restricted than in microbial systems.

“…The purified leucine tRNA isoaccepting species were assayed as to their cytokinin content by gas chromatographic methods previously described (3). It was found that leucine isoaccepting species I, V, and VI showed hydrolysis products which migrated in the positions of standard cytokinin-like compounds.…”

Section: Rpc -2mentioning

confidence: 99%

Purification of Leucine tRNA Isoaccepting Species from Soybean Cotyledons

Lester

Morris²,

Cherry³

1979

Self Cite

Two of the six leucine isoaccepting tRNA species from soybean (Glycine max) cotyledons recognize U-ginning codons, and contain cytokinin moieties in their structure. These same two isoaccepting species have been shown to undergo quantitative changes in their relative amounts upon treatment with N-benzyladenine in vivo. In addition a procedure has been developed for purification of the isoaccepting species of leucine tRNA from soybean cotyledons resulting in isoacceptors of minimum purity, calculated by amino acid acceptance capacity, of from 46 to 78% leucine tRNA.The idea that tRNA has a regulatory function in the translation of mRNA originated with Ames and Hartman (1) and was expanded and modified by others (2, 13-15). The salient feature of this proposal was that the rate of synthesis of certain proteins was limited not by the supply of the appropriate mRNA but by the supply of specific tRNAs required to read "rate-limiting" or minor codons present in the message.A cytokinin is present in the structure of several tRNA isoaccepting species. Because the mechanism of hormonal action of the cytokinins is not understood one can propose that the cytokinin molecule is involved in the activity of specific isoaccepting tRNA. The native cytokinin is adjacent to the anticodon of specific tRNAs from mycoplasma to man (12). In addition, it has been shown that all known cytokinin-containing tRNA species translate codons with the initial nucleotide U (8, 12). It seems clear from previous studies (5) that the presence and integrity of the N6-A2 (isopentenyl) adenosine adjacent to the anticodon is essential for proper binding to the messenger ribosome complex and thus of importance for protein synthesis.After purification of various isoaccepting species of leucine tRNA present in soybean some fundamental questions concerning the connection between these tRNAs and cytokinin might be answered. For