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. 2007 Mar 26;104(14):5788–5793. doi: 10.1073/pnas.0606880104

Fig. 1.

Fig. 1.

Preparation and hydrodynamic behavior of 40S complexes (see SI Materials and Methods for full details). (A) eIFs, 40S, and 40S-MFC (43S) complex loaded onto an SDS/PAGE gradient gel. A few of the eIF proteins show very similar electrophoretic behavior to 40S proteins and thus generate multiple bands (marked by asterisks) in the 43S lane. The 43S complex loaded here was isolated as fractions (see vertical dotted lines in B) from a sucrose-density gradient. (B) A 254-nm absorbance elution profile from a sucrose-density gradient; the presence of the eIFs in the 43S sucrose-gradient peak was routinely confirmed by means of Western blotting using the antibodies defined in SI Materials and Methods. (C) The 40S (gray, circles) and the 43S complex (red, diamonds) were subjected to sedimentation velocity analytical ultracentrifugation, yielding apparent sedimentation coefficient distributions. These were fitted with Gaussian equations (lines), giving sedimentation coefficients of 36.4S for the small subunit and 41.5S for the preinitiation complex. Corrected for finite solute and solvent effects on the basis that the small subunit value is 40S, the preinitiation complex value becomes 45.6S. (D) Assembly of complexes between 40S and MFC eIFs. PhosphorImages of native gels loaded with 40S–eIF complexes incorporating [35S]Met-tRNAi and GMP-PNP. The addition of eIF3 leads to a shift in the ribosome complex band (left-hand side). The further addition of eIF5B and the 60S subunit led to a further shift, associated with the formation of 80S (right-hand side). (E) A typical experiment following the kinetics of methionyl-puromycin synthesis by 80S complexes prepared by using the components shown in A plus 60S, eIF5B, methionyl-tRNAi, and GTP (instead of GMP-PNP). A PhosphorImage of samples from a typical experiment that were allowed to run on cation exchange TLC is shown. (F) Time course of formation of the 35S-labeled methionyl-puromycin product, taking the samples featured in E plotted against time. The y axis is calibrated in relative signal intensity. Also plotted are data from an experiment in which eIF5B was omitted.