Electron microscopic determination of the binding sites of ribosomal proteins S4 and S8 on 16S RNA.

Specific complexes.early in the assembly of Escherichia coli ribosomes were examined in the electron microscope. Complexes between ribosomal protein S4 or S8 and 16S RNA were fixed gently with formaldehyde and then denatured for protein-free spreading. Binding of each protein was found to preserve an easily recognized configuration in the RNA that allows the sites of protein binding to be determined. S8--16S RNA complexes have a single hairpin loop near the middle of the 16S RNA, 798 +/- 21 bases from one end and 657 +/- 26 bases from the other. S4-16S RNA complexes have two adjacent loops at one end with 250--450 bases. This structure probably arises from the simultaneous binding of S4 to three noncontiguous sites on the RNA. Measurements of these complexes place the binding sites near the 5' end, at more than one site 250--585 nucleotides from the 5' end and 645 +/- 45 bases from the 3' end. The latter site has not been recognized previously as a distinct S4 binding site. This approach allows the binding sites to be determined without knowledge of the nucleotide sequence and gives insight into the configuration of the rRNA in the assembling ribisome.