Isolation and Characterization of Two Methionine: tRNA Ligases from Wheat Germ

Abstract

Two methionine: tRNA ligases (here called ligase A and ligase B) with distinctly different enzymatic and molecular properties were isolated in homogeneous form from extracts of raw wheat germ. Both the A and B enzyme are composed of single polypeptide chains of M , 105000 and 70000 respectively. The smaller molecule (B) has been shown not to be a proteolytic fragment of the larger one (A). The catalytic properties of both the A and B enzymes have been established and the Mg2+-dependent capacity to charge six purified methionine-accepting tRNAs have been compared to those of the methionine: tRNA ligases from Escherichia coli and bakers' yeast. The possible reasons for the presence of two methionine: tRNA ligases and their unusual monomeric nature are discussed.Methionine : tRNA ligase is of particular interest in the study of ligase-tRNA interactions for a variety of reasons. First, most, if not all, of the methionine : tRNA ligases catalyze the aminoacylation of both the homologous initiator and elongator methionine tRNAs. Since tRNAp' and tRNAE' serve such different roles in protein synthesis, the common structural features that account for recognition by the same ligase should be more easily defined. Second, the covalent structure of the enzyme from Escherichia coli and Bacillus stearothermophilus is being actively studied [1,2] and the discovery of internal repeating sequences has posed some interesting functional and evolutionary questions. Third, proteolysis of the enzyme from E. coli [3] and from B. stearothermophilus [4] results in a shortening of the protomer and disruption of the naturally occurring oligomers leaving an enzymatically active monomeric fragment. Crystals of the active fragment of the E. coli methionine : tRNA Abbveviations. BD-cellulose, benzyolated DEAE-cellulose; t R N A y and tRNAP' indicate methionine-accepting tRNAs that function as initiator tRNAs in protein biosynthesis but which are distinguished by the ability ( i e . , tRNAr") or the inability (ie., tRNAPt) of their methionyl adducts to act as substrates for the E. roti transformylase. tRNA,M"' denotes those methionine-accepting tRNAs that participate in the elongation stage of protein synthesis.Definitions. One unit of enzymatic activity is that amount of enzyme that catalyzes the incorporation of 1 nmol methionine into methionyl-tRNA in 10 min under the conditions specified in Materials and Methods. ,4280 unit ( A 2 5 8 unit), the quantity of material contained in 1 ml of solution which has an absorbance of 1 at 280 nm (258 nm) when measured in a 1-cm path-length cell.Enzyme. Methionine: tRNA ligase (EC 6.1.1.10).ligase produce excellent X-ray diffraction patterns and are currently under study [ 5 ] .The crystallographic structure determination of yeast tRNAp' is presently under way in this laboratory [6]. In conjunction with this study we undertook to purify methionine: tRNA ligase from a variety of sources, particularly eukaryotes. Here we report the purification and molecular properties of two methionine : tRNA ligases f...

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

“…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] A-50 were purchased from Pharmacia Pine Chemicals. D~AE~e~~o~ @E-23) was a product of ~a~~~ ~ospho~~u~o~ was obtained from Serva Company.…”

“…wt 173 000) and other MetRS f14,15]. According to [6] wheat germ contains two MetRS ligases called A and B with mol. wt 105 000 and 70 000, respectively.…”

The 3 A crystal structure of yeast initiator tRNA: functional implications in initiator/elongator discrimination.

“…Preparation of crystals Yeast tRNA,Met was purified from crude brewer's yeast tRNA, purchased from Boehringer Mannheim, essentially as described (Rosa and Sigler, 1977) except that the tRNAs were eluted isochratically off the Sepharose4B column in 1.6 M (NH4)2SO4, 10 mM sodium acetate, 10 mM MgCI2, pH 4.5. Crystals were grown by the vapor diffusion technique and stabilized essentially as described (Schevitz et al, 1979).…”

Two initiation sites detected in the small s1 species of reovirus mRNA by dipeptide synthesis in vitro.

“…Artemia salina cysts were purchased from San Francisco Brand (Newark, CA). Escherichia coli tRNAASP, tRNA~Iu, tRNA2Yr, tRNA la, tRNA aI, and tRNA1er were purchased from Subriden RNA (Rolling Bay, WA) and yeast tRNAet was initially obtained from P. Sigler (University of Chicago, IL) and later prepared according to a modification of a published procedure (16). Unfractionated E. coli tRNA was from Sigma, and crude brewers yeast tRNA was from Boehringer Mannheim.…”