Significance of the third tRNA binding site, the E site, on E. coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non‐cognate aminoacyl‐tRNA to the A site

• Published in: The EMBO journal 1990-12, Vol.9 (13), p.4527-4533
• Main Authors: Geigenmüller, U., Nierhaus, K. H.
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Binding, Competitive; Escherichia coli; Escherichia coli - genetics; Protein Biosynthesis; Reproducibility of Results; Ribosomes - chemistry; Ribosomes - metabolism; RNA, Transfer - metabolism; RNA, Transfer, Amino Acyl - metabolism
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1990.tb07904.x

The E site (exit site for deacyl‐tRNA) has been shown to be allosterically linked to the A site (aminoacyl‐tRNA binding site), in that occupation of the E site reduces the affinity of the A site, and vice versa, whereas the intervening peptidyl‐tRNA binding site (P site) keeps its high affinity. Here the question is analysed of whether or not the low affinity state of the A site caused by an occupied E site is of importance for the ribosomal accuracy of the aminoacyl‐tRNA selection. In a poly(U) dependent system with high accuracy in poly(Phe) synthesis, the acceptance of the cognate ternary complex Phe‐tRNA–EF‐Tu–GTP (which has the correct anticodon with respect to the codon at the A site) was compared with the competing acceptance of ternary complexes with near‐cognate Leu‐tRNA(Leu) (which has a similar anticodon) or non‐cognate Asp‐tRNA(Asp) (which has a dissimilar anticodon), by monitoring the formation of AcPhePhe, AcPheLeu or AcPheAsp, respectively. Cognate (but not near‐cognate) occupation of the E site reduced synthesis of the ‘wrong’ dipeptide AcPheLeu only marginally relative to that of the cognate AcPhe2, whereas the formation of AcPheAsp was decreased as much as 14‐fold, thereby reducing it to the background level. It follows that the allosteric interplay between E and A sites, i.e. the low affinity of the A site induced by the occupation of the E site, excludes the interference of non‐cognate complexes in the decoding process and thus reduces the number of aminoacyl‐tRNA species competing for A site binding by an order of magnitude.