UC Santa Cruz

Translocation of mRNA unidirectionally through the ribosome is coupled to subunit rotation. Based on our observation that A1503 of 16S rRNA intercalates between mRNA bases during specific rotational states, we tested the possibility that it could play a role in maintenance of the reading frame. We eliminated the possibility of intercalation by creating ribosomes containing an abasic site at position 1503 of 16S rRNA. This was done by site-specifically cleaving and sequentially ligating a synthetic RNA oligonucleotide containing abasic 1503. While directing ligation of the synthetic RNA to cleaved 16S rRNA, we were able to preserve higher-order structure by using a tailed DNA splint which could be removed quantitatively under mild conditions. Reconstituted ribosomes containing the abasic site were fully active in protein synthesis but showed an increased level of spontaneous frameshifting. In testing our control ligation constructs that did not contain abasic 1503, we established that semi-synthetic 16S rRNA has slightly elevated rates of frameshifting. We confirm that this finding is attribute to the demethylated 16S rRNA bases A1518 and A1519, which are universally methylated in natural ribosomes.