3'-terminal polyadenylate sequences of Escherichia coli tryptophan synthetase alpha-subunit messenger RNA.

Our earlier studies have shown that the mRNA from many bacterial species, including Escherichia coli and Bacillus subtilis, is extensively polyadenylated, but with shorter poly(A) segments than those associated with eukaryotic mRNA. In this paper, we show that about 40% of the mRNA for the tryptophan synthetase alpha-subunit (TrpA) of E. coli carries a 3'-terminal polyadenylate sequence of 15 to 20 residues. This conclusion was supported by several independent lines of evidence. About 40% of trpA mRNA bound to oligo(dT)-cellulose at high ionic strength and was eluted with water. Treatment with RNase H in the presence of oligo(dT)12-18 destroyed the ability of trpA mRNA to bind to oligo(dT)-cellulose, presumably through the degradation of the poly(A) tract. trpA mRNA could be used as template for complementary DNA synthesis with reverse transcriptase in a reaction that was absolutely dependent on oligo(dT)12-18 as primer. The identity of the cDNA product as a complement to trpA mRNA was established by specific hybridization. In addition, it was possible to synthesize polyadenylated trpA mRNA in toluene-permeabilized cells of E. coli transformed with a recombinant plasmid carrying the trpA gene. In view of the fact that the trpA gene and its 3'-untranslated region contain no continuous deoxyadenylate sequences larger than five nucleotides, one can conclude that the polyadenylate moiety is added post-transcriptionally.