Crosslinking of Escherichia coli initiation factor IF-3 to the RNA moiety of the 30S ribosomal subunits

Abstract

Escherichia coli initiation factor IF-3 labeled in vitro by either reductive alkylation or reaction with TV-ethylmaleimide can be cross-linked to the 16S rRNA of 30S ribosomal subunits by irradiation with ultraviolet light or reaction with formaldehyde, respectively. In both cases, the cross-linking to the 16S rRNA appears to be direct and not mediated via ribosomal proteins. When cross-linking to 16S and 23S rRNAs is compared at short irradiation times or under conditions which depress nonspecific IF-3 binding, the factor is cross-linked two to three times more efficiently to the 16S than to the 23S rRNA. When cross-linked complexes of 30S and radioactive IF-3 are isolated and subjected to limited hydrolysis with RNase Tl, the 30S ribosomes are split into two subparticles which can be electrophoretically resolved; the two particles each contain a characteristic set of proteins with one The topographical localization of the IF-3 binding site on the 30S ribosomal subunit has been attempted by different techniques. The picture emerging from these studies is that, when bound to the ribosome, IF-3 is localized in a region probably adjacent to the subunit interface (Gualerzi et al., 1973) neighboring, at least in part, the 3' end of the 16S rRNA (van Duin et al., 1975) as well as ribosomal proteins SI, S7, SI 1,

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

“…Two discrete crosslinks between IF-3 and 16S rRNA in situ are in agreement with these findings (33)(34)(35) (33)(34)(35) as well as electron microscopic visualization of the factor on the 30S subunit (37) allow for fairly precise localization ofthe binding site for IF-3 on the platform side of the cleft of the 30S subunit. The tertiary structure model for the arrangement of rRNA in the 30S subunit (3) is consistent with this localization, since it places the 3'-end crosslink region (33)(34)(35), the crosslink region of position 840 (35), and the region of position 791 of this study in the same general area of the 30S subunit. The clustering of these rRNA regions on the platform at the interface between 30S and 50S subunits is consistent with the functional relationships between IF-3 and 30S subunits during initiation complex formation.…”

Cross-Linking of Proteins to Nucleic Acids by Ultraviolet Light

“…The former complex has the tRNA localized in the A site of the ribosome, while the tRNA is in the P site in the latter complex_ They found that a number of proteins from both the 30S subunit and the 50S subunit were bound to the tRNA when localized in either the A site or the P site. Pon et al (1977) found that the translation initiation factor IF-3 could be bound by UV irradiation to the 16S RNA of the E. coli 30S ribosomal subunit; less efficient UV-induced binding to the 23S RNA of the 50S subunit could also be observed. It was determined that the cross-linking was mainly associated with one of two RNase Tl fragments resulting from limited hydrolysis of the irradiated ribosome-IF-3 complex by this enzyme.…”

Structure-function relationship in Escherichia coli initiation factors: role of tyrosine residues in ribosomal binding and functional activity of IF-3

“…When the iodination is completed, the IF-3 activity is lost by 90%, as determined by its capacity to promote the dissociation of ternary complexes of poly(U)-N-AcPhe-tRNA-30S ribosomal subunits. The capacity of IF-3 to bind to the 30S ribosomal Initiation factor IF-3 binds to the 30S ribosomal subunit by interacting with the 16S rRNA (Gualerzi & Pon, 1973;Pon & Gualerzi, 1976;Pon et al, 1977). Since the primary sequence of IF-3 is known (Brauer & Wittmann-Liebold, 1977) and the identification of the region of the rRNA to which IF-3 binds is in progress, it would be desirable to obtain some information concerning the nature and the number of active sites of this important component of the protein synthetic machinery.…”