Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.
Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D003597:Cytosine Nucleotides; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012343:RNA, Transfer / Q000096:biosynthesis*; D012260:Ribonucleases; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
D003597:Cytosine Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; D011117:Polyribonucleotide Nucleotidyltransferase; D005092:Exonucleases; D012260:Ribonucleases