Isolation and sequence determination of 5'-terminal oligonucleotide fragments of RNA transcripts synthesized by bacteriophage T₃-induced RNA polymerase from T₃ DNA

Abstract

The nucleotide sequence of the 5'-terminal oligonucleotides produced by pancreatic RNase digestion of bacteriophage T3 RNA polymerase (EC 2.7.7.6) transcripts of T3 DNA has been determined. The sequence determination is based upon a simple isolation procedure for the 5'-terminal oligonucleotides. This procedure involves treatment of pancreatic RNase digests of α ³²P-labeled T3 RNA polymerase transcripts with bovine brain exoribonuclease to remove oligonucleotides with free 5'-hydroxyl termini and then chromatographing the products on hydroxylapatite to resolve the remaining oligonucleotides having 5'-phosphate termini. By application of standard two-dimensional separation and sequence techniques, the major 5'-end sequences deduced were pppGpGpGpApGpApGpApY(Y = pyrimidine nucleoside) and pppGpGpGpApGpApCp. In addition, the sequences of other minor 5'-terminal oligonucleotides observed on homochromatograms were also determined. The sequences of these 5'-oligonucleotides were pppGpGpGpApApCpY, pppGpGpGpApApUpY, pppGpGp(2-4 Gp, 2-3 ApGp)..., and pppGpGpGp.... These results demonstrate that T3 phage-induced RNA polymerase possesses a high degree of specificity in the initiation of RNA chains.