During polypeptide elongation cycle, prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs. Continued progress has been achieved in understanding this key process, including results of structural, ensemble kinetic and single-molecule studies. However, most of work has been focused on the pre-equilibrium states of this fast process, leaving the real time dynamics, especially how EF-G interacts with the A-site tRNA in the pretranslocation complex, not fully elucidated. In this thesis, the kinetics of EF-G catalyzed translocation is investigated by both ensemble and single molecule fluorescence resonance energy transfer studies to further explore the underlying mechanism. In the ensemble work, EF-G mutants were designed and expressed successfully. The labeled EF-G mutants show good translocation activity in two different assays. In the smFRET work, by attachment of a fluorescent probe at position 693 on EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. The EF-G:L11 FRET efficiency is constant during the entire EF-G occupancy on the ribosome, while the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. Transition from intermediate to low is the rate-limiting step with a small FRET change, whereas transition from high to intermediate is fast, showing a large FRET change. In total, we capture a ~12 Ã? relative movement of A-site tRNA away from EF-G during translocation. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. This EF-G:tRNA smFRET measurement was applied to bifunctional rhodamine labeled EF-G derivatives (BR-EF-G) and comparable kinetic results were obtained. FRET changes of BR-EF-G:Cy5-tRNA pair are larger if the BR group is located closer to the EF-G hinge region, suggesting that parts of domain IV of EF-G, which penetrate to the decoding region of the ribosome, move together with the A-site tRNA during translocation.