EFFICIENT IN-VITRO TRANSLATIONAL TERMINATION IN ESCHERICHIA-COLI IS CONSTRAINED BY THE ORIENTATIONS OF THE RELEASE FACTOR, STOP SIGNAL AND PEPTIDYL-TRANSFER-RNA WITHIN THE TERMINATION COMPLEX

There have been contrasting reports of whether the positioning of a tr anslational stop signal immediately after a start codon in a single ol igonucleotide can act as a model template to support efficient in vitr o termination. This paradox stimulated this study of what determines t he constraints on the positioning of the components in the termination complex, The mini mRNA, AUGUGAA, was unable to support efficient in v itro termination in contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the stop signal with the decoding factor, re lease factor 2, was stimulated with ethanol or with nucleotide-free re lease factor 3, or by using (L11(-))-ribosomes which have a higher aff inity for release factor 2, or unless the fMet-tRNA was first bound to 30S subunits independently of the mini mRNA, An additional triplet st op codon could restore activity of the mini mRNA, indicating that its recognition was not sterically restrained by the stop signal already w ithin it, This suggests that in an initiation complex an adjoining sta rt/stop signal is not positioned to support efficient decoding by rele ase factor unless it is separated from the start codon. Site-directed crosslinking from mRNAs to components of the termination complex has s hown that mRNA elements like the Shine-Dalgarno sequence and the codon preceding the stop signal can affect the crosslinking to release fact or, and presumably the orientation of the signal to the factor.