Effect of bases contiguous to AUG on translation initiation.

We report that bases 3’ to AUG have a much smaller effect on the formation of initiation complexes than do bases on the 5’ side. This relative lack of effect was shown to hold under conditions which stringently require initiation proteins for complex formation. The following oligonucleotides formed initiation complexes as efficiently as did AUG: AUGA, AUGG, AUGU, AUGC, AUGAUG, AUGGCU, AUGUCU, A U G W , AUGULJA, and AUGAGC. In contrast, a more efficient formation of a 30 S o initiation complex occurred with the UAUG uersus RAUG or AUGN tetramers. This was reflected in an enhanced rate of complex formation. All complexes formed were stable at 0 ”C. At 24 “C, the complexes exhibited slow first order dissociation kinetics in dilute solution. All AUGN-programmed complexes exhibited similar rates of reversibility under these conditions. The preference for 5’ pyrimidines was observed with a series of pentamers and hexamers of varied base composition. This discrimination of context by the 30 S subunit took place in the presence or absence of initiation factors. Since UAUG does not directly bind to met-tRNA, but AUGA does, the data suggest that the anticodon of met-tRNA is altered during formation of the initiation complex. The rate and extent of met-dipeptides synthesized with UAUGU3 was higher than with AAUGU,, or with hexamers containing AUG followed by A, U, or G, e.g. AUGAUG, AUGGCU, AUGUUU, and AUGUUA, underscoring the importance of the 5’ uridine in stimulating initiation.