Figure 2.

Stepwise translation of the MFK-encoding mRNA analogue. (A) Partial sequence of the mRNA analogue. The Shine–Dalgarno sequence, the codons being translated and the corresponding toe-print stops are marked. The toe-print stop is repeatedly appearing at the nucleotide +16 relative to the 5′-most nucleotide at the P-site. (B) Toe-print analysis of the stepwise translation progress. ‘WT’ and ‘C2394G’ mark the complexes formed by wild-type ribosomes and ribosomes carrying the C2394G mutation, respectively. Lane 1, complex of ribosomes (0.1 µM), MFK-mRNA (0.1 µM) and ${\text{tRNA}}_{\text{f}}^{\text{Met}}$ (0.2 µM). Lane 2, ribosomes (0.1 µM), MFK-mRNA (0.1 µM), ${\text{tRNA}}_{\text{f}}^{\text{Met}}$ (0.2 µM) and AcPhe-tRNA^Phe (0.2 µM). Lane 3, same as in lane 2 but after the addition of EF-G*GTP (0.1 µM). Lane 4, corresponds to lane 3 but with Lys-tRNA^Lys*EF-Tu*GTP (0.2 µM) added. Reverse transcriptase stops are marked by numbers starting from the A in the initiation AUG codon. The primer was hybridized 50 nt downstream of the AUG codon.