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. 2012 Mar;18(3):508–518. doi: 10.1261/rna.027698.111

FIGURE 8.

FIGURE 8.

Filter binding analysis of tRNAfMet dissociation from 70S ternary complexes with 5′-phosphate or 5′-hydroxyl LL cI-lacZ mRNA. Ternary complexes are formed with mRNA (0.5 μM), 30S subunits or 70S ribosomes (0.7 μM), and 32P-labeled tRNAfMet (≤0.05 μM). Reactions were diluted 100-fold into buffer containing 400-fold excess unlabeled tRNAfMet. Samples were spotted onto a double membrane system over time (bottom). The nitrocellulose upper membrane shows tRNAfMet bound to ribosomes (i.e., tRNA in ternary complex) and the positively charged lower membrane shows tRNAfMet not in ternary complex (i.e., free tRNA). The graph shows the proportion of tRNAfMet bound to ribosomes after phosphorimaging quantification of radiolabeled tRNA bound to each membrane and is used to derive the complex stabilities for radiolabeled tRNA in ternary complexes containing LL cI-lacZ mRNA with a 5′-phosphate (LL 5′P) or a 5′-hydroxyl (LL 5′OH), or ternary complexes containing SDL cI-lacZ mRNA with a 5′-phosphate (SDL 5′P).