IDENTIFICATION OF RIBOSOMAL-PROTEIN S1 AS A POLY(A) BINDING-PROTEIN IN ESCHERICHIA-COLI

To elucidate the metabolic function of mRNA polyadenylation in Escheri chia coli, we searched for a polyadenylate-binding protein as a potent ial mediator of the function of the poly(A) moiety. Using a nitrocellu lose filter-binding assay and a Northwestern blot technique, a protein in the ribosomal supernatant fraction of E coli was identified and pu rified to homogeneity. N-terminal sequence analysis yielded a 25-resid ue sequence which corresponded to the 25 N-terminal amino acids of pro tein S1, one of the proteins of the E coli 30S ribosomal subunit. Poly (A) binding to S1 protein was inhibited by Mg2+ and Mn2+ and by ATP an d stimulated 8-fold by 100 mM KCI. The binding of S1 to poly(A) occurr ed with an association constant of 3 x 10(6) M-1 and seemed to be only mildly cooperative. Competition studies of the binding of poly(A) and poly(C) to purified S1 protein were consistent with the presence of t wo polynucleotide binding sites, of which one binds poly(A) five times more strongly than poly(C), whereas the other binds poly(C) 50 times more strongly than poly(A). Poly(A) bound to 30S ribosomal subunits bu t not to 50S ribosomes. To study possible association of S1 with the p oly(A) tracts of E coli mRNA in the proces of translation, poly(A) RNA was isolated from polysomes by oligo(dT) cellulose chromatography and the poly(A) RNA with bound protein was eluted either directly or afte r digestion with RNase T1 and A. When subjected to Western blot analys is with antibody to S1, both poly(A) RNA and isolated poly(A) tracts r evealed bound S1 protein. The implications of these results for the po ssible interaction of poly(A) tracts of mRNA and the translational mac hinery of E coli are discussed.