Ribosomal affinity and translational initiation in Escherichia coli: In vitro investigations using translational initiation regions of differing efficiences from the atp operon

Abstract

The atp operon of Escherichia coli comprises nine genes that are differentially expressed. The control of the atp genes' expression rates has been shown to be exercised primarily at the level of translational initiation, but how is this achieved in molecular terms? In order to study the interactions of 30 S ribosomal subunits with specifically the translational initiation regions (TIRs) of atpB, atpE and atpG, restriction fragments bearing these TIRs were excised from the atp operon and cloned into an SP6 promoter transcription vector. mRNA transcripts were made in vitro and used in primer extension inhibition studies and equilibrium mRNA-30 S ribosomal subunit binding measurements. The binding of 30 S ribosomal subunits blocked primer extension 14 to 15 bases downstream from the respective translational start codons. The affinities of binding of 30 S ribosomal subunits showed the relationship atpE > atpB > atpG. This was also the order of the efficiency of translation promoted by the respective TIRs, both in vivo and on the in vitro synthesized mRNA fragments. Thus, the affinity of 30 S ribosomal subunits is at least to some extent correlated with the rate of translational initiation.