Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E. coli and yeast. Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases. Leucyl-tRNA synthetase from E. coli shows a high initial substrate discrimination. Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs. Lactonisation of gamma-hydroxyleucine and homocysteine could be detected. Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level. No lactonisation nor mischarged tRNALeu is detectable.

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP) : tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6 and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP) : tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described. ATP(CTPj : tRNA nucleotidyl transferase catalyzes the incorporation of CMP and AMP into tRNA lacking the pCpCpA part of its 3' terminus [l]. Whereas tRNA with an incomplete 3' end cannot participate in any step of the elongation cycle during protein biosynthesis, after regeneration of the CpCpA end it regains its full biological activity. ATP(CTP) : tRNA nucleotidyl transferase can also be used for the preparation of modified tRNA species since it has been shown that some analogues of ATP [2 -51 and CTP [6,7] are also substrates for this enzyme. The tRNA species with an altered CpCpA terminus thus formed can be used for investigations of the mechanisms of aminoacylation [8 -121, ribosomal protein synthesis [9,13-151 as well as for spectroscopic [16,17] and X-ray crystallographic [18,19] studies.Tn the course of our investigations a considerable number of ATP and CTP analogues were tested as PO-

ABSTRACTtRNAPhe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates. Out of several tRNAPhe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNAPheC-C-2'dA and tRNAPheC-C-formycinoxi-reds are strong competitive inhibitors for tRNAPheC-C-A during phenylalanylation. In the ATP/PPi exchange, both these inhibitors reduce Vman, to about 25%o; but whereas tRNAPheCC_2'dA has no influence on Km ATP and Km Phe during ATP/PPj exchange, tRNAPheCC-formycinox i-red reduces Km AT! from 1430 IAM, found in the absence of tRNAPhe, to 230 jiM, and Km Phe, from 38 to 14 uM. The values found in the presence of tRNAPheC-C-CformycinOxi-re during ATP/PPi exchange, are identical with those determined in the phenylalanylation of tRNAPheC_C-A.All other tRNAPhe species carrying a modified 3'-end that cannot be phenylalanylated exhibit a mixed competitivenoncompetitive inhibition in the phenylalanylation reaction. In the ATP/PPi exchange, they do not influence Km ATP and K,, Phe and only weakly, if at all, Vmax.The results show that the 3'-adenosine of tRNAPhe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction. The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNAPhe triggers a conformational change of the enzyme.In the living cell part of the genetic information stored in the DNA is translated into proteins. This is achieved by first transcribing the information into mRNAs and then translating the latter into peptide sequences on the ribosome. Prior to translation on the ribosome, the amino acids are activated by attachment to the 3'-terminal ribose of their particular tRNAs.The overall aminoacylation reaction is achieved according to tRNAaa + ATP + aa + E 2 aa-tRNAaa + AMNP + PPj + E, where E is the enzyme and aa is the amino acid. This reaction can be followed experimentally by the incorporation of radioactively labeled amino acid into tRNA.

Formycinmonophosphate (FMP) has been incorporated in to the 3'-terminus of phenylalanine tRNA from yeast by tRNA nucleotidyl transferase. tRNAPhepCpCpF can be aminoacylated with the cognate synthetase exhibiting the same K , and an about 50-times lowered V compared to tRNAPhepCpCpA. Quantum yield of FMP fluorescence in tRNAPhepCpCpF is about four times lower than in the monomer. Comparative studies of tRNAPhepCpCpF and the oligonucleotide CpApCpCpF show that the differences between the fluorescence properties of tRNAPhepCpCpP and the monomeric formycin-nucleotide are due only t o stacking interactions and do not arise from the tertiary structure of tRNA. After periodate oxidation of the 3'-terminal ribose of tRNAPhepCpCpF the full fluorescence of formycin is reconstituted. This emphasises the important role of the intact ribose ring for stabilization of stacking units. Periodate oxidation of tRNAPhepCpCpF followed by borohydride reduction leads t o a tRNA that acts as competitive inhibitor in the aminoacylation reaction. This, together with its high quantum yield makes it a valuable tool in fluorescence spectroscopic studies of the aminoacylation reaction.

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.