THE IDENTITY OF THE BASE FOLLOWING THE STOP CODON DETERMINES THE EFFICIENCY OF IN-VIVO TRANSLATIONAL TERMINATION IN ESCHERICHIA-COLI

A statistical analysis of >2000 Escherichia coli genes suggested that the base following the translational stop codon might be an important feature of the signal for termination. The strengths of each of 12 possible 'four base stop signals' (UAAN, UGAN and UAGN) were tested in an in vivo termination assay that measured termination efficiency by its direct competition with frameshifting. Termination efficiencies varied significantly depending on both the stop codon and the fourth base, ranging from 80 (UAAU) to 7% (UGAC). For both the UAAN and UGAN series, the fourth base hierarchy was U > G > A similar to C. UAG stop codons, which are used rarely in E.coli, showed efficiencies comparable with UAAN and UGAN, but differed in that the hierarchy of the fourth base was G > U similar to A > C. The rate of release factor selection varied 30-fold at UGAN stop signals, and 10-fold for both the UAAN and UAGN series; it correlated well with the frequency with which the different UAAN and UGAN signals are found at natural termination sites. The results suggest that the identity of the base following the stop codon determines the efficiency of translational termination in E.coli. They also provide a rationale for the use of the strong UAAU signal in highly expressed genes and for the occurrence of the weaker UGAC signal at several recoding sites.