Polyriboadenylate Polymerase and its Relationship with RNA Polymerase

Abstract

POLYRIBOADENYLATE polymerase¹ is a ubiquitous enzyme whose biological role is not fully understood. Recently, the inhibition of poly A polymerase in Escherichia coli infected with T-even phages² was tentatively related to the concomitant arrest of the synthesis of macromolecule in the bacterial host³. Its function might be further elucidated if a conditional lethal mutant could be found in which poly A polymerase is temperature sensitive. Screening for such a mutant among a set of ts mutants unable to perform protein synthesis at high temperature was undertaken by Dr G. Tocchini-Valentini using techniques already described⁴. 1 ml. of tryptone broth containing 5 × 10⁸ cells of any one of the mutant derivatives of E. coli D₁⁴ (Hfr C met⁻RC^rel) was sonicated and mixed with 1.5 ml. of 20 mM Tris (pH 9.5), 4.9 g/l. of magnesium acetate, 20 mg/l. deoxyribonuclease (ribonuclease free, Sigma), 10 mg/1. tRNA (from E. coli, General Biochemical), 55 mg of ¹⁴C-ATP (New England Nuclear) at a specific radioactivity of 0.5 mCi/mmole. This was done in duplicate: one preparation was then incubated for 20 min at 30° C, and the other at 42° C. Incubation was stopped by the addition of an equal volume of 10 per cent trichloroacetic acid (TCA) and the samples were filtered on ‘Millipore’ filters and counted in a gas flow low-background counter. To check that the TCA insoluble product from this crude extract was really poly A, a control was performed using a-32P-ATP, hydrolysing the precipitate with alkali and checking that all the radioactivity was recovered after paper electrophoresis (in acetate buffer, pH 3.5) in the AMP spot. In a set of 200 ts mutants unable to synthesize protein at high temperature, three exhibited temperature sensitive poly A polymerase activity. They were crossed and gave no wild type recombinants. Successive experiments were done using one of the three which we call C134 (see Table 1).