The ribosome is the large molecular machine that uses an mRNA template to synthesize proteins, in a process referred to as translation. The functions of the ribosome can be separated into four phases, which are initiation, elongation, termination of protein synthesis, and the recycling phase. At the heart of translation is the elongation cycle. This process is catalyzed by two guanosine triphosphatase (GTPase) factors, elongation factors Tu (EF-Tu) and G (EF-G). During translocation, EF-G catalyzes the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) inside the ribosome, coupled to intricate conformational rearrangements of the machinery1,2. How EF-G initiates translocation and why it consumes guanosine triphosphate (GTP) are key questions unresolved. Here, we analyze the importance of the highly conserved loops I and II at the tip of EF-G domain IV and demonstrate that loops I and II play a crucial role in translocation and particularly residues of loop II are essential for triggering the process. Our results provide evidence that the decoding-center (DC) interactions with the codon-anticodon duplex set the threshold for translocation. EF-G catalyzes translocation by releasing the duplex from the DC with its highly conserved loops I and II at the tip of domain IV, and hence inducing subsequent translocation actions of 30S swiveling and tRNA2•mRNA movement. However, GTP hydrolysis on EF-G is irresponsible for translocase activity, but needed by the factor for its multiple-turnover.