Eur. J. Biochem. 187, 543-548 (1990) zyxwvuts 8 FEBS 1990 zyxwvutsrqpo The ribosomal domain of the bacterial release factors The carboxyl-terminal domain of the dimer of zyxwvu Eschevichia coli ribosomal protein L7/L12 located in the body of the ribosome is important for release factor interaction Warren P. TATE', Berthold KASTNER', Christina D. EDGAR', Kim K. McCAUGHAN', Kirsten M. TIMMS', Clive N. A. TROTMAN', Marina STOFFLER-MEILICKE', Georg STOFFLER', Bishwajit NAG3 and Robert R. TRAUT3 zy ' Microbiology Institute of the Medical Faculty, University of Innsbruck, Innsbruck, Austria Department of Biochemistry, University of Otago, Dunedin, New Zealand Department of Biological Chemistry, School of Medicine, University of California, Davis, USA (Received July 3/0ctober 9, 1989) - EJB 89 0820 1. Polyclonal antibodies (pAb 1 - 73 and pAb 26 - 120) have been raised against both an N-terminal fragment of zyxwvutsrqp Escherichia coli ribosomal protein L7/L12 (amino acids 1 -73), and a fragment lacking part of the N-terminal domain (amino acids 26 - 120). 2. Only pAb 26 - 120 inhibited release-factor-dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C-terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3. A monoclonal antibody against an epitope of the C-terminal two thirds of the protein (mAb 74-120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4. A monoclonal antibody against an epitope of the N-terminal fragment of L7/L12 (mAb 1 - 73), previously shown to remove the dimer of L7/L12 in the 50s subunit stalk but still bind to the body of the particle, partially inhibited release-factor-mediated events. 5. The mAb 74- 120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1 - 73, indicating that the body L7/L12 dimer, and in particular its C-terminal domains, are important for release factor/ribosome interaction. 6. The two release factors have subtle differences in their binding domains with respect to L7/L12. Ribosomal protein L12 or its N-terminally acetylated de- rivative L7 is found in four copies on the ribosome [l], and most probably in situ it is present as two dimers [2], a form which exists in solution [3]. L7/L12 has been the only protein mapped by electron microscopy to the stalk structure which extends from one side of the subunit [4, 51. Recently monoclonal antibodies against two epitopes at the N- and C-terminal parts of the protein have been mapped by immunoelectron microscopy [6]. The antibody against the N-terminal epitope caused the detachment of one dimer and the disappearance of stalks. Antibodies were still bound on the body of the stalkless subunits [7], whereas the antibody against the C-terminal epitope had two distinct binding sites, the distal tip of the stalk and the body of the subunit from where the stalk projects. The results were explained by a model in which one dimer of L7/L12 exists folded on the subunit body while the second is extended in the flexible subunit stalk (61. Correspondence to W. P. Tate, Department of Biochemistry, Uni- versity of Otago, P.O. Box 56, Dunedin, New Zealand Ahbreviutions. RF-I , release factor-I ; RF-2, release factor-2; pAb, polyclonal antibody; mAb monoclonal antibody; EF-Tu, elongation factor Tu; EF-G, elongation factor G; 1-73, 1-120,26-120, 74- 120, amino acid residues 1 -73, 1 ~ 120,26- 120,74- 120 respective- ly, of the L7/L12 ribosomal protein L7/L12 has been shown to be important for release-factor- mediated events from studies where L7/L12 was removed by ethanol ammonium chloride [S] or using polyclonal antibodies against the whole protein [9]. In the former case however, the release factor could still function but with a much reduced binding affinity [lo]. At this time it seemed that L7/L12 in the stalk might provide an initial docking site for the release factors before they moved into a functional site within the interface between the two ribosomal subunits. L11 has also been shown to be a critical determinant nearby for release factor binding [l 1 - 131 and therefore this protein was thought to provide the binding domain of the functional site on the large subunit for the release factor molecules. In the current study the region of L7/L12 important for release factor func- tion and its location to the stalk or body of the large subunit of the ribosome have been investigated. MATERIALS AND METHODS Preparation ojribosomes and release factors 70s ribosomes and their subunits were prepared from Escherichia coli MRE 600. Ribosomes were isolated as tight couples although in some cases the final gradient step was omitted [14]. E. coli t R N A p was aminoacylated with [3H]methionine and formylated as described [15]. Release