@article{9f14c2078d5c42c8955f4f57fded8719,
title = "Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit",
abstract = "Complexes of 30 S subunits and [14C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.",
author = "Heimark, {Ronald L.} and Lawrence Kahan and Kathleen Johnston and Hershey, {John W.B.} and Traut, {Robert R.}",
note = "Funding Information: Certain proteins cross-linked to initiation factors have also been identified as cross-linked pairs in experiments on the free 30 S subunit: S13-S19 (Lutter e,t al.. 1974; Sun et al., 1974a); S14-819 (Bode et al., 1974); and S7-S13, 511-513, S12-S13 and S12-S21 (Sommer & Traut. unpublished results). A trimer of X11, S18, 521 has also been reported (Shih & Craven, 1973). One of the antibody combining sites of 54 is located near S13 (Fig. 5(b)) : a cross-linked pair containing S4 and S13 has been reported (Sommer & Traut, 1974). Other experimental evidence is relevant to the work reported here. Hawley ef al. (1974) reported that IF3 was covalently cross-linked with DMS to the 30 S protein S12. one of the proteins identified here. Protein S7: not, found in the present study, was shown to be cross-linked to IF3 by the reagent tart,aryl diazide (Van Duin et al.: 1975). Our failure to detect an S7-IF3 cross-link with PDM is explicable by the lack of cysteine residues in this protein (Kahan et al., 1974; Moore, 1975). The failure to form such a cross-link with DMS may be related t,o t,he differing lengths and reactivities of the cross-linking agents. Inhibition of IF3 binding has been observed by pre-incubation of 30 S subunits with antibodies aga,inst probeinx S4, S7, S12, 513. S14 and S19 (Gualerzi & Pan: 1973). These results are largel,v consistent with t)he cross-linking results. St,udies using the periodate-oxidized 3{\textquoteright} terminus of 16 S RNA as an RNA-protein cross-linking derivative have shown the covalent attachment to RNA of IF3 (Van Duin rt aZ.. 1975) and of proteins Sl and S21 (Kenner, 1973; Czernilofsky ef al., 1975). It has also been shown t{\textquoteright}hat the binding of the 30 S proteins S7, S9, S13 and S19 is localized near the 3{\textquoteright} end of the 16 S RNA (Zimmerman et al., 1974). Three of these prot{\textquoteright}eins have been shown by cross-linking to be involved in the initiation site. This region of IF3 binding is interesting in relation to the factor{\textquoteright}s role in mediating the forma,tion of the mRNA-30 S complex (Vermeer rt al., 19733). Recently it has been shown that the 3{\textquoteright} end of the 16 S RNA can be isolated in a base-paired complex wit,h all initiator region of mRN{\textquoteright}$ from a, preformed initiation complex (Steitz & Jakes. 1975). Heterologous reconstitution experiments wiith the 30 S protein 512 isolat{\textquoteright}ed from Bacillw stearothwm,ophilus have shown bhat protein 512 and the 16 S RNA are responsible for the recognition of initiation signals (Held et al., 1974; Goldberg $ St*eit,z, 1974). A chemically reactive analog of the initiation codon AUG was found t,o label four of t{\textquoteright}he proteins identified here : Sll, S12, S13 and S21, in addition to S4 and S18 (Pongs et al.. 1975). Furt{\textquoteright}hermore, poly(s41J), an affinity a,na{\textquoteright}log of poly(U). was found to covalently hind to proteins Sl. S18 and S21 (Fiser of ad.. 1975). The results summarized above are all consistent with the involvement of proteins Sl. S4, ST, Sll, S12. S13, S18, X19 and S21 in a region near the 3{\textquoteright} end of the 16 S RNA and in the region of mRNA binding. The data presented here indicate that six of t,hese proteins (Sl, Sll, SlP. 813: S19 and S21) are directI!: or indirectly crosslinked t,o IF3 and suggest{\textquoteright} that the binding sit{\textquoteright}rs for initia,tion fa,ct{\textquoteright}ors and mRNA are highly overlapping. \Vo t,llartk Dr M. Nomura for advice and support during the cowso of this work and Dr W. Benisek for providing DMS, MS J. Yanov for the preparation of IF3 and Mr S. Langbrrg and Mr J. Kenny for critical reading of the manuscript. This work was supported by grants from the United States Public Health Service (GM-17924 t)o K. R. T., GM-15422 to Dr M. Nomura and GM-i2150 to L. K.), from t,he American Cancer Society (NP-70 to .J. W. R. H.) and from the National Science Founds-tion ((:I3-31086X to Dr M. Nornl~tx).",
year = "1976",
month = aug,
day = "5",
doi = "10.1016/0022-2836(76)90108-X",
language = "English (US)",
volume = "105",
pages = "219--230",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",
}