Reassembly in vitro has been used to prepare a set of twenty Escherichia coli 30 S ribosomes containing pairs of proteins carrying different covalently attached fluorescent labels. These 30 S particles have normal sedimentation properties and moderate to excellent activity in polyphenylalanine synthesis. Reassembly of the dye-conjugated proteins proceeds well but exhibits a preference for incorporation of species containing fewer dyes than the average of the population. Singletsinglet energy transfer measurements have been carried out on all samples. The majority of the results are highly reproducible. A wide range of efficiencies is seen for protein pairs and the results sort roughly into four proximity classes. Assuming a Poisson distribution of dye stoichiometries and either random surface labeling on spherical proteins or specific unique labeling sites, observed efficiencies were analyzed to yield distance estimates for each protein pair. These were also generally quite reproducible for different samples of a given protein pair. The distance estimates show that four protein pairs are so close that extensive, protein-protein contact is likely. Six others are close enough so that these proteins could well be nearest neighbors in the 30 S particle although there is probably intervening RNA. The remainder are far enough apart that other proteins or extensive regions of the 16 S rRNA are likely to lie in between. There is sufficient distance information on six proteins to conclude that their three-dimensional arrangement has a marked deviation from planarity. Variations in transfer efficiency as a function of the degree of labeling indicate that S20 protein has a length at least twice what would be expected for the equivalent spherical protein. All of the distance estimates available are highly consistent with other known proximity information. The detailed results suggest that proteins linked by reassembly in vitro are usually close but are not necessarily in direct contact. This would mean that many of the assembly steps are mediated by the 16 S rRNA.
ASJC Scopus subject areas
- Molecular Biology