Isolation and Properties of tRNA Nucleotidyl Transferase from Yeast

Abstract

tRNA nucleotidyl transferase has been purified 5000‐fold from commercial bakers' yeast. The isolation procedure includes ammonium sulfate fractionation and chromatography on DEAE‐cellulose, CM‐cellulose, Biogel and phosphocellulose. The enzyme is homogeneous on analytical centrifugation and by sodium dodecylsulfate gel electrophoresis. The molecular weight of the native and denatured enzyme Mc=Or is 71000 as determined by ultracentrifuge measurements and 70000 as determined by sodium dodecylsulfate gel electrophoresis. On micro isoelectric focusing the enzyme shows an isoelectric point at about pH 7.5. The enzyme catalyses the incorporation of ATP and CTP into the 3′‐end of tRNA, and, in the presence of pyrophosphate, the pyrophosphorolyses of adenylic and cytidylic acid residues from the 3′‐end of tRNA.

Incorporation of 5‐Iodocytidine into Yeast tRNAPhe with tRNA Nucleotidyl Transferase in vitro

“…Snake venom phosphodiesterase (1 mg/ml) was a product of Boehringer Mannheim GmbH (Mannheim, Germany). tRNA nucleotidy1 transferase obtained from different steps during purification from bakers' yeast [6] was used. Its specific activity was a t least 4000 units/mg ; 1 unit = incorporation of 1 nmol ATP into tRNAPhe-A,,-C,,-C,,/min.…”

“…The ~RNAP~~-A,,-C,,-~O~C,~-A~~ and tRNAPhe-A,,-io5C,,-io5C,,-A7,, respectively, thus obtained can be aminoacylated by the corresponding synthetase exhibiting the same K , but a slightly lowered V compared to tRNAPhe-A73-C74-C75-A, 6. An analytical method is presented which enables quantitative determination of the 3' terminal nucleosides.…”

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

“…The details and results of the purification of the yeast methionine tRNAs are given in Fig.1 and Table 1. Following the purification of the yeast tRNAE', the 3'-terminal sequence was completed [16] using CTP, ATP and yeast nucleotidyl transferase [25]. The specific activities of the methionine tRNAs (nmol methionine accepted/Azss unit) were : yeast tRNAp" 1.8; yeast tRNAE' 1.5; wheat germ tRNAp' 1.8; wheat germ tRNAz" 1.3; E. coli t R N A p 1.9; E. coli tRNAF' 1.3.…”

Isoleucyl‐tRNA synthetase from baker's yeast: The 3′‐hydroxyl group of the 3′‐terminal ribose is essential for preventing misacylation of tRNAIle‐C‐C‐A with misactivated valine

“…The assay mixture contained 150 mM TrisHCl buffer pH 7.6,150 mM KCl, 10 mM MgSO*, 0.06 mM ['4C]isoleucine or [*4C]valine and 0.003 mM tRNAUe-C-C-N. The amount of isoleucyl-tRNA synthetase (EC 6.1.1.5) of specific activity 552 units/mg [8] and valyl-tRNA synthetase (EC 6.1.1.9) of specific activity 276 unitslmg [8] is given in table 1.20 E.cg of tRNA-nucleotidyl transferase (EC 2.7.7.21) of specific activity 5000 units/mg [9] in the 100 fl assay mixture were used where specified.…”