initiator tRNA analogs with different nucleotides in the

The ef fec t of base changes a t the fourth pos i t ion from the 3 ' terminus of Esoheriohia c o l i i n i t i a t o r tRNA has been s tudied to t e s t the ' d i s criminator hypothesis ' which proposed tha t the nucleot ide in t h i s pos i t ion might have a r o l e i n the s p e c i f i c i t y of the aminoacylation r eac t ion . £ . ool i i n i t i a t o r tRNA lacking the 3 ' t e rmina l t e t ranuc leo t lde was prepared by p a r t i a l d iges t ion with S1 nuclease. To const ruct tRNA analogs with different bases in the fourth pos i t ion t h i s t runcated tRNA was Joined by RNA l igase to each of four chemically synthesized 2 ' ,3 ' -ethoxy-methylidene te t r anuc leo t ides pACCA(em), pCCCA(em), pGCCA(em), and pDCCA(em). In v i t r o aminoacylation s tud ies showed tha t a l l four molecules accepted methionine, albeit with different V values. max INTRODUCTION The 'tRNA recognition problem', the question of what specific nucleotides in a tRNA molecule determine the specificity of the aminoacylation reaction (1,2), has fascinated biochemists for a long time. Ten years ago the 'discriminator hypothesis' was proposed, to subdivide the family of tRNAs into groups for recognition purposes (3). It was suggested that the nucleotide occupying the fourth position from the 3'-terminus of a tRNA could serve as a primary discriminator. Each such group would possess its own recognition code; tRNAs with similar base sequence but changes in the discriminator nuoleotide may be charged with different amino acids. In the intervening years a large number of tRNA species from many different organisms have been sequenced (4). Comparison of their sequences shows that the discriminator nucleotide is generally, but not exclusively, conserved within an isoacceptor family in the same organism. In addition, mutant tRNA species with changes in the discriminator nucleotide are known, which possess different amino acid specificity (5,6). Recent advances in the methodology of chemical and enzymatic synthesis of ribo-oligonucleotldes have allowed the construction of relatively large RNA molecules. For instance, RNA species with the nucleotide sequence © I R L Press Limited, Oxford, England. 6531 0305-1048/82/1020-6531S 2.00/0 Nucleic Acids Research Met of £. coll initiator tRNA (7) or yeast alanlne tRNA (8) have been constructed by joining chemically synthesized oligoribonucleotides with RNA llgase, and many different yeast tRNA analogs (9) have been prepared by an enzymatic procedure (10). These approaches lend themselves to site directed mutagenesis of tRNA species, which should be useful in unraveling the molecular details of the functions of these molecules. E.. coli initiator tRNA possesses an adenosine in the discriminator position. He decided to construct analogs of this tRNA which contain different bases in the fourth position from the 3'-terminus (position 74) to determine the effect on aminoacylation by methionyl-tRNA synthetase. This was accomplished by joining with RNA ligase chemically synthesized tetranucleotides of the structure pNCCA to an initiator tRNA fragment lacking the terminal tetranucleotide. MATERIALS AND METHODS General. T4 RNA ligase was purified as described earlier (11). Polynucleotide kinase and £. coll alkaline phosphatase were gifts of M. Suglura. ( -P)ATP was prepared by the method of Walseth and Johnson (12). &. sail. tRNA*J was a gift of Dr. A. Kelmers (Oak Ridge National Laboratory) and had a specific acceptor activity of 1.4 nmol/A26 unit. Polyacrylamide gel electrophoresis was carried out in the presence of 7 M urea (13). Oligonucleotldes. 2',3'-Ethoxymethylidene tetranucleosides of the structure pXCCA(em) were synthesized by first Joining of p -adenosine 5»-p -2',3'-ethoxymethylideneadenosine, A(5')ppA(em), to trinucleoside diphosphates NpCpC (N=A,G,U and C) with RNA ligase (11) and then introducing a 5'-phosphate using polynucleotide kinase (15) and ( -P)ATP of specific activity (50 cpm/pmole). S1 Met Nuolease S1 digestion of tRNA. £. colj. initiator tRNA (100 units) in 2 ml of 1 mM ZnCl 5 mM MgCl was digested with nuclease (3X10 units) at 37° for 2 hr. After phenol extraction, the partially digested tRNA was precipitated with ethanol (6 ml) and the RNA mixture separarated by polyacrylamide (10J) gel electrophoresis. The desired product, tRNA lacking the 3'-terminal tetranucleotide and designated tRNA(S1), was isolated in about 30$ yield. Mat* Perlodate oxidation a£. tRNA. £. sslX. tRNATM (60 A2g() units in 3 ml) was treated with 9 mM sodium periodate in 0.1 M sodium acetate (pH 5.2) at 37° for 4 hr. The solution was then made 0.5 M in lysine-HCl (pH 8.0) to cause base elimination (16). The oxidized tRNA was then treated with