The iterative movement of the tRNA-mRNA complex through the ribosome is a hallmark of the elongation phase of protein synthesis. We used synthetic anticodon stem-loop analogs (ASL) of tRNA(Phe) to systematically identify ribose 2'-hydroxyl groups that are essential for binding and translocation from the ribosomal A site. Our results show that 2'-hydroxyl groups at positions 33, 35, and 36 in the A site ASL are important for translocation. Consistent with the view that the molecular basis of translocation may be similar in all organisms, the 2'-hydroxyl groups at positions 35 and 36 in the ASL interact with universally conserved bases G530 and A1493, respectively, in 16S rRNA. Furthermore, these interactions are also essential for the decoding process, indicating a functional relationship between decoding and translocation.
