A major internal initiation site for the in vitro translation of the adenovirus DNA polymerase.

An open reading frame which encodes at least 90% of the adenovirus type 2 DNA polymerase gene was cloned behind the SP6 promoter and transcribed in vitro using the SP6 RNA polymerase. The resultant RNA was translated in a rabbit reticulocyte cell free system. In addition to the translation of a 120-kDa protein corresponding to the size of the complete open reading frame, the synthesis of a 62-kDa polypeptide was demonstrated. Data is presented to show that the synthesis of the 62-kDa polypeptide resulted from internal initiation of translation in frame in the middle of the message at the 11th or 12th AUG. Capping of the mRNA resulted in an increase in synthesis of the 120-kDa protein and a concordant decrease of the internally initiated polypeptide. We propose that there may be competition between the binding of the translational preinitiation complex at or near the 5' end of the mRNA and at the internal initiation site. Because of inhibition of synthesis of the 120-kDa but not the 62-kDa polypeptide by hybrid arrested translation using DNA complementary to approximately one third of the 5' Ad Pol mRNA sequences, scanning of the ribosome from the 5' end of the mRNA to the internal initiation site seemed unlikely. The sequence proximal to the 12th AUG is ACCCACCCCAUG which is similar to a noncontinuous sequence 5'AUCCACC(X)nAUG complementary to the 3' end of the 18 S rRNA. This sequence is a favored ribosome binding site based on the observation that it is the most commonly observed one at or near the 5' end of 162 mRNA's analyzed (D. R. Sargan, S. P. Gregory, and P. H. W. Butterworth, 1982, FEBS Lett. 147, 133-136).