The induction of tRNA synthesis following T4 phage infection

Abstract

T4 bacteriophage infection of E. coli B cells induces the formation of 4S RNA molecules that specifically hybridize with T4 DNA. The T4 4S RNA extracted from the hybrid was found to contain pseudouridylic acid, suggesting that some of this RNA might have amino acid acceptor function. In order to study the amino acid acceptor capacity of the T4 4S RNA, two procedures were devised. The first one involved the isolation and purification of specific 4S RNA-DNA hybrids in a manner that avoided the use of RNase and permitted the extraction of biologically active tRNA from the hybrid. It was found that a significant fraction of the T4 4S RNA isolated by this method had amino acid acceptor activity. This was shown by assaying with a mixture of 15 14C-labeled amino acids or with [14C]leucine alone. In the second method, T4 N-acetyl[3H]aminoacyl-tRNA was prepared in order to stabilize the aminoacyl-tRNA ester bond. T4 N-acetyl[3H]leucyl-tRNA was incubated with T4 DNA in the presence of 50% formamide at 30°C. Sephadex G-200 chromatography revealed that a significant fraction of the N-acetyl[3H]leucyl-tRNA hybridized with the T4 DNA. Another procedure involved the hybridization of N-acetyl[3H]-aminoacyl-tRNA with T4 DNA at 70°C in a citrate buffer in the absence of formamide. The annealing mixture also contained a 20-fold excess of uncharged E. coli tRNA. The hybrid-containing solution was loaded onto nitrocellulose filters and treated with T1 RNase. At this point, the T4 tRNA was found to contain leucine-, arginine-, isoleucine-, and possibly tyrosine-specific chains.