M. Barciszewska

Purification and properties of methionyl-tRNA-synthetase from yellow lupine seeds

In many euca~~tic org~isms studied so far there are two rne~iQ~e.~~~i~c tRNAs: initiator (tRNAy ) and non-initiator (tRNAr). The primary structures of several taco have been elucidated and the e~ste~~e of their great similarities ~tab~~ed_ Roth tRNAfet are recognized by the same rne~ouy~tRNA ~ntheta~ @ietRS, EC 6.l.l.lO) [l--3]. Purification and properties of MetRS from wheat germ have been published and differences haw; appeared [4-61. In our studies on plant tRNA and aminoacyltRNA synthetases we have isolated tRNA?t and MetRS from yellow Iupine seeds. In work on rne~on~e-tRNA hgase we have found some interesting obse~a~ons ~o~~e~~g pu~~~tio~ and properties of this enzyme. The procedure for isolator consists of the ~~5~~~1 s~~hate fractionation, gel f&ration on Sephadex G-150, DEAEcellulose, DEAE-Sephadex A-50, Sephadex G-ZOO filtration and phosphocellulose column chromatography, We have obt~Ed a 545-fold puri~cation of the enzyme with the recavery of over 1.6% activity applied In the first step. Polyacryhunide gel electrophoresis and gel fdtration method showed mol, wt 170 000, The yeflow lupine MetRS has two subunits and the results took similar to those obtained for wheat germ /4,5] but different from [6] _

Higher plant 5S rRNAs share common secondary and tertiary structure. A new three domains model.

A new model of secondary and tertiary structure of higher plant 5S RNA is proposed. It consists of three helical domains: domain alpha includes stem I; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. Except for, presumably, a canonical RNA-A like domain alpha, the two remaining domains apparently adopt a perturbed RNA-A structure due to irregularities within internal loops B and E and three bulges occurring in the model. Bending of RNA could bring loops B and E and/or C and D closer making tertiary interactions likely. The model differs from that suggested for eukaryotic 5S rRNA, by organization of domain gamma. Our model is based on the results of partial digestion obtained with single- and double-strand RNA specific nucleases. The proposed secondary structure is strongly supported by the observation that crude plant 5S rRNA contains abundant RNA, identified as domain gamma of 5S rRNA. Presumably it is excised from the 5S rRNA molecule by a specific nuclease present in lupin seeds. Experimental results were confirmed by computer-aided secondary structure prediction analysis of all higher plant 5S rRNAs. Differences observed between earlier proposed models and our proposition are discussed.

Conservation of the structures of plant tRNAs and aminoacyl-tRNA synthetases

Since the determmatlon of the first nucleotlde sequence of a transfer rlbonuclelc acid (tRNAAh) in 1965 [l], almost 200 primary structures of other tRNAs have been determined [2,3] Among them tRNAs specific for phenylalanme and methlonme are of most interest The former for their easy punticatlon by benzoylated DEAE-cellulose column chromatography [4] and the latter m respect to their special functions m protein biosynthesis [5]. Both tRNAs were Isolated from different sources [6] and some slmllarltles appeared At the same time many ammoacyl-tRNA synthetases from different orgamsms were punfied and some generahsatjons concerning their structures have been formulated [7,8]. Among large number of organisms from which tRNAs and synthetases were characterized,, higher plant material was little studied, one reason being difficulty m preparation of sufficient quantities of these macromolecules m the pure state [9,10] As far as tRNA 1s concerned, phenylalanme specific tRNA was sequenced from wheat germ [ 111, pea [ 121, yellow lupm [ 131 and barley [ 141 Methlonme mltlator tRNA structure was determined for wheat germ [ I.51 and tentatively, yellow lupine seeds [ 161 Ammoacyl-tRNA synthetases (AARS) charging cognate tRNA with phenylalanme (PheRS), methlonme (MetRS), argimne (ArgRS) and leucme (LeuRS) from wheat germ [ 17-191 and yellow lupm seeds [3-O-22] were characterized. The purpose of this work 1s to compare and analyse the sumlarltles and differences m the structures of

Effect of elastase on elongation factor 1 from wheat germ

Abstract Elongation factor 1, species A, B and C, were isolated from wheat germ and purified to homogeneity by the following steps: supernatant 100 000 xg, precipitation with ammonium sulphate and column chromatography: Sephadex G-150, DEAE-Sephadex A-50 and hydroxylapatite. On the second column the activity was divided into three peaks: EF1 A, B and C. The pure proteins EF1A, B and C (molecular weight 61 000, 48 000 and 12 500 D, respectively) were treated with elastase. Two products of EF1A digestion, polypeptides b and c, were isolated. The molecular weights of polypeptides b and c were similar to molecular weight of species B and C of EF1. Both digestion products were active in binary complex formation with GDP and in binding of Phe-tRNA to ribosomes. EF1B was converted to polypeptide c or similar and EF1C was rather resistant to elastase treatment.

The initiator transfer ribonucleic acid from yellow lupin seeds, correction of nucleotide sequence and crystallization

Abstract Two methionine specific tRNAs from yellow lupin seeds have been purified to homogeneity. Initiator tRNA (tRNA i Met ) but not tRNA m Met was charged with Escherichia coli methionyl-tRNA synthetase. The nucleotide composition, T i and pancreatic RNase digestion fingerprints and nucleotide sequence of lupin tRNA i Met showed its identity with wheat germ and bean initiator tRNAs. The differences in the primary structure of the lupin tRNA i Met observed by other authors have not been confirmed. We have defined the conditions under which single crystals of lupin tRNA i Met can be grown reproducibly.

Lack of cytokinin activity of Y-type bases isolated from phenylalanine specific tRNAs

Abstract This work describes the lack of cytokinin activity of Y-type bases. These results clarify contradictory results existing in the literature.

The dynamic conformation of plant cytoplasmic 5S rRNAs

Recently we have proposed a new three-dimensional model of plant 5S rRNAs structure. To verify this proposal we present here new data on RNase T1 digestion and hydroxyl radical hydrolysis of lupin and wheat germ 5S rRNAs at various buffer and temperature conditions. Interestingly, the guanosine residues 85-87 in the loop D of these RNAs, are resistant to RNase T1 at native but not at denaturating conditions. On the other hand, the reaction of 5S rRNA with the hydroxyl radicals showed different reactivity of many nucleotides in various parts of the molecule and suggest conformational changes, which occur mostly in the loops. The experimental data clearly support involvement of the nucleotides occupying conserved positions in the loops in the tertiary interactions in plant 5S rRNA structure.

Unfolding of the tertiary structure of specific tRNA and ribosomal 5S RNA from plants as studied with hydroxyl radicals

Ribosomal 5S RNA is present in all eubacterial and eukaryotic ribosomes. Despite a large amount of experimental data on the primary and secondary structures of these types of molecules, details of their tertiary structure and their precise function in protein biosynthesis are still not known. Recently we have proposed a new model for the tertiary structure of plant 5S rRNA. In this study we applied the Fe(II)-mediated cleavage reaction to test the model. The data presented here provide experimental evidence that in the 5S rRNA molecule only a few nucleotides are buried in the tertiary structure. Similar experiments performed with methionine initiator tRNA gave results which imply the difference in its structure when compared with the X-ray structure of yeast tRNAPhe.

Interaction of alkaloids with plant transfer ribonucleic acids. Effect of sparteine on lupin arginyl-tRNA formation

The effect of the alkaloid sparteine on arginyl-tRNA formation was studied. It was demonstrated that sparteine sulfate in the concentration range 10-60 mM inhibits the charging reaction when amino acid, ATP and tRNA are used as variable substrates. The mode of action is different for all pattern of inhibition for all varied substrates is generally uncompetitive. A pattern of inhibition for all varied substrates is generally uncompetitive. A non-competitive mechanism for amino acid and tRNA was observed at low sparteine concentration, but in the case of ATP it is also uncompetitive.

Method for isolation of aminoacyl-tRNA synthetases from plants: purification and some properties of methionyl, phenylalanyl and arginyl tRNA synthetases from yellow lupin seeds

Abstract A method for the simultaneous purification of methionyl-, phenylalanyl- and arginyl-tRNA synthetases from yellow lupin seeds ( Lupinus luteus ) is described. The method uses ammonium sulphate fractionation, and DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. Molecular weight and kinetic parameters of the pure enzymes are reported.