Studies on the environment of protein S7 within the 30-S subunit Escherichia coli ribosomes

Abstract

Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 AND S19, together with RNA from sections O'-D-E'-K-P-P'-E-A of the 16-S sequence. 1. When 30-S subunits were hydrolysed with ribonuclease T1 in presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections O'-E' and P-A of the 16-S RNA, but that section K (from the middle of this area) was missing. 2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified. It was possible to demonstrate that the site of cross-linking lies within the O'-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region. 3. When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences, but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections O'-E' and P-A, but that section K was missing (cf. first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein.