Ribosomes from E. coli, f[3H]Met-tRNAfMet, [14C]Phe-tRNAPhe, [14C]Lys-tRNALys, Glu-tRNAGlu, Gly-tRNAGly, Phe-tRNAPhe, Val-tRNAVal, BODIPY-[3H]Met-tRNAfMet, initiation factors, and EF-Tu were prepared as described (27, 35, 36). The gene coding for EF-G was cloned into pET24 with a C-terminal histidine tag, and mutations were introduced by site-directed mutagenesis. All mutations were verified by DNA sequencing. Wt EF-G and EF-G mutants were overexpressed in BL21 (DE3) cells and purified using Protino Ni-IDA (Macherey-Nagel) in gravity flow columns as described (37). All mRNAs were synthesized by IBA Lifesciences (Göttingen). Start codons are in bold, and slippery site are underlined. The following mRNA sequences were used to study reading frame maintenance: 5′-GUUAACAGGUAUACAUACUAUGGAAAAGUUCAUUACCUAA-3′, 5′-GUUAACAGGUAUACAUACUAUGGGAAAGUUCAUUACCUAA-3′, 5′-GGGAAUUGUGAGCGGAUAACAAUUCCCCUCUAGAGCAGUUGCAGCGCGUGCAGGGAGCAACCAUGGCAAAAAAGUUCUAG-3′, and 5′-GGGAAUUGUGAGCGGAUAACAAUUCCCCUCUAGAGCAGUUGCAGCGCGUGCAGGGAGCAACCAUGGCAAAGAAGUUCUAG-3′.

For the translocation experiments, the following mRNA was used:


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