RNA ligase catalyzed the joining of pC‐C‐Ap with C‐A‐A in the synthesis of C‐A‐A‐C‐C‐Ap, which has the sequence of the Escherichia coli tRNAMetf 3′‐end. pC‐C‐A was also shown to be joined to C‐A‐A without any undesired self‐polymerization. Joining of pC‐C‐A to various synthetic ribotriplets, such as C‐C‐A, A‐A‐A, C‐C‐C, U‐U‐U, U‐A‐G, C‐C‐G and U‐U‐C, was performed as well as joining to the partially substituted trimers with a photolabile o‐nitrobenzyl group, C‐Anbzl‐A and C‐C‐Anbzl. The yields were C‐A‐A‐C‐C‐A (69%), C‐C‐A‐C‐C‐A (38%), A‐A‐A‐C‐C‐A (66%), C‐C‐C‐C‐C‐A (71%), U‐U‐U‐C‐C‐A (50%), U‐A‐G‐C‐C‐A (23%), C‐C‐G‐C‐C‐A (43%) and U‐U‐C‐C‐C‐A (46%). C‐Anbzl‐A was a slightly poorer acceptor than C‐A‐A and C‐C‐Anbzl did not serve as an acceptor. Recognition of acceptor molecules by RNA ligase is discussed in terms of affinity of oligonucleotides for the enzyme.

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E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.

. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [ Y -~~P ] ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural t R N A p from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11 % and 84% with respect to that of the intact tRNA7'.

A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNA; except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths.of3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase andjoined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.Chemical synthesis ofnucleic acids has been a challenging problem in organic chemistry since the structure ofthe nucleic acids was elucidated. Chemical methods to synthesize short ribo-and deoxyribopolynucleotides with defined sequences were established in early 1960s, and those oligonucleotides were important in the elucidation of the genetic code (1). Discovery of DNA ligase allowed the synthesis of bihelical DNAs from chemically synthesized deoxyribopolynucleotides. With this chemical-enzymatic method the genes for yeast alanine tRNA (2) and Escherichia coli tyrosine tRNA precursor (3) have been synthesized; the latter was the first synthetic functional DNA molecule. Genes for peptides have also been synthesized by the same approach, and the methods for joining double-stranded DNA pieces with protruding ends have been used in various recently developed reactions for genetic manipulations.Although tRNAs are the smallest nucleic acids with unique functions, their synthesis has been difficult until recently, mainly because of the lack ofgood synthetic methods for larger .oligoribonucleotides as well as a lack ofjoining enzymes. After the primary structure of yeast alanine tRNA had been determined (4), the nona-and hexanucleotide corresponding to the terminal sequence of this tRNA were synthesized by phosphodiester block condensation. These fragments in turn were used to form reconstituted molecules with natural tRNA fragments derived by RNase digestions. However, aminoacylation was not possible because the synthetic fragments were too small to form sufficiently stable complexes for recognition by the alanyl-tRNA synthetase (5). The discovery of RNA ligase (6) and its ability to join single-stranded oligoribonucleotides (7) made it possible to elongate synthetic RNA fragments to yield larger molecules such as tRNAs.The initiator methionine tRNA of prokaryotes has a special role in protein biosynthesis, which. manifests itself in several unique properties of that tRNA (8). It was also the subject of detailed modif...