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

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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 was determined. The sequence determination is based on a simple isolation procedure for the 5''-terminal oligonucleotides. This procedure involves treatment of pancreatic RNase digests of .alpha.-32P-labeled T3 RNA polymerase transcripts with bovine brain exoRNase 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 2-dimensional separation and sequence techniques, the major 5''-end sequences deduced were pppGpGpGpApGpApGpApY (Y = pyrimidine nucleoside) and pppGpGpGpApGpApCp. 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.... T3 phase-induced RNA polymerase possesses a high degree of specificity in the initiation of RNA chains.