Anticodon loop mutations perturb reading frame maintenance by the E site tRNA

Abstract

The ribosomal E site helps hold the reading frame. Certain tRNA mutations affect translation, and anticodon loop mutations can be especially detrimental. We studied the effects of mutations saturating the anticodon loop of the amber suppressor tRNA, Su7, on the ability to help hold the reading frame when in the E site. We also tested three mutations in the anticodon stem, as well as a mutation in the D stem (the “Hirsh” mutation). We used the Escherichia coli RF2 programmed frameshift site to monitor frame maintenance. Most anticodon loop mutations increase frameshifting, possibly by decreasing codon:anticodon stability. However, it is likely that the A site is more sensitive to anticodon loop structure than is the E site. Unexpectedly, the Hirsh mutation also increases frameshifting from the E site. Other work shows that mutation may increase the ability of tRNA to react in the A site, possibly by facilitating conformational changes required for aminoacyl-tRNA selection. We suggest that this property may decrease its ability to bind to the E site. Finally, the absence of the ms2io6A nucleoside modifications at A37 does not decrease the ability of tRNA to help hold the reading frame from the E site. This was also unexpected because the absence of these modifications affects translational properties of tRNA in A and P sites. The absence of a negative effect in the E site further highlights the differences among the substrate requirements of the ribosomal coding sites.