Studies of the GTPase domain of archaebacterial ribosomes

Abstract

Ribosomes from the methanogens Methanococcus vunnielii and Methanobacterium jormicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichiu coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolohus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L 11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L 11 from E. coli bound well to 23 S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.Few functional domains of the eubacterial ribosome have been well characterized but one such is the GTPase centre whose activity is coupled to elongation factor G (EF-G). Much of the information concerning this active site has been gathered by studying the interaction of the inhibitor thiostrepton with the larger (50 S) ribosomal subunit or with subparticles derived from it (for review, see [I]). Thus, the primary binding site for thiostrepton has been localized to 23 S rRNA within the region where protein L11 of the 50s ribosomal subunit also interacts [2]. Binding of the drug to 23s rRNA is relatively weak ( K d approximately 0.5 pM; M. Stark and E. Cundliffe, unpublished data) but is dramatically enhanced when protein L 11 is also complexed with the RNA. As a result of such binding to native ribosomes, thiostrepton specifically inhibits factor-dependent GTP hydrolysis [3]. Further evidence that protein L l l participates in the ribosomal GTPase domain was forthcoming when this protein was labelled within the ribosome by a photoactivated derivative of GTP, in a reaction dependent upon the presence of factor EF-G [4]. Also, when factor EF-G was cross-linked to 70s ribosomes, protein L11 was again one of the targets [5], as was that specific portion of 2 3 s rRNA which is protected by protein L11 [ti].In addition to its involvement in GTPase activity, the domain of the 50s ribosomal subunit with which thiostrepton interacts also plays a role in the regulatory coupling of translation to a whole array of other cellular processes including the synthesis of rRNA, tRNA, ribosomal proteins and enzymes involved in amino acid biosynthesis (for review, see [7]). This occurs via the so-called 'stringent response' and involves the interaction of stringency factor with the aforementioned ribosomal domain. In response to the binding of uncharged tRNACorrespondence to E. Cundliffe, Department of Biochemistry, University of Leicester, Leicester, England LE 1 7 RH Abbreviations. Mc2S0, dimethylsulphoxide; EF-G, elongalion factor G ; EF-2, elongation factor 2; SDS, sodium dodecyl sulphate; ppGpp, guanosine 5'-diphosphate 3'-diphospha...

The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA

“…Fig. 5A) is involved in EF-G-dependent functions (5)(6)(7)(8)(9)(10)(11). Thiostrepton, an antibiotic that inhibits binding of EF-G to ribosomes (5), binds to a 23S rRNA fragment that has A1067 (26); in addition, methylation of the 2Ј-hydroxyl of A1067 confers resistance to thiostrepton in strains of Streptomyces aureus (6).…”

“…There are threedimensional structures of EF-G alone (3) and in a complex with GDP (4) from x-ray crystallography; however, little is known of the chemistry of the binding of the factor to ribosomes. There is evidence for the interaction of EF-G with two separate sites in Escherichia coli 23S rRNA: with the thiostrepton region (5)(6)(7)(8)(9)(10)(11) and with the sarcin͞ricin (S͞R) domain (11,12).…”

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

“…This is comparable with the fact that rat L12 and anti-28 S recognize a similar tertiary structure of the eukaryotic GTPase domain. The E. coli-L8 complex (a counterpart of the rat P complex) binds to the GTPase domain of 23 S rRNA through L10 moiety (Pettersson et al, 1976;Pettersson, 1979;Beauclerk et al, 1984) and protects mainly the internal loop region comprising residues 1044 -1050 and 1109 -1111 against chemical reagents and nuclease (Egebjerg et al, 1990;Rosendahl and Douthwaite, 1993). This loop region is highly conserved; 7 of 10 bases are preserved between E. coli and rat.…”

“…This RNA region has been identified as a site of interaction with elongation factor G (Sköld, 1983;Moazed et al, 1988) and with the antibiotic thiostrepton, an inhibitor of elongation factor-dependent processes in protein synthesis (Thompson et al, 1982;Egebjerg et al, 1989). Ribosomal protein L11 and the acidic protein complex L10(L12) 4 cooperatively bind to this RNA domain (Dijk et al, 1979;Beauclerk et al, 1984) and construct a functional site of the 50 S subunit. L11 may participate in induction of a functionally important RNA conformation (Xing and Draper, 1995).…”

Serological association of lupus autoantibodies to a limited functional domain of 28S ribosomal RNA and to the ribosomal proteins bound to the domain

“…With this method, we demonstrated that a human autoantibody specifically binds to an RNA region corresponding to residues 1944-2002 of human 28S rRNA (or residues 1767 1825 of mouse 28S rRNA) [9]. This region is highly conserved and termed the GTPase domain [9,16]. To address questions of the frequency of such autoantibody in patients with SLE and of whether 28S rRNA has any other autoimmune targets, sera from 120 patients (90 patients with SLE, 10 with rheumatoid arthritis, five with scleroderma, four with polymyositis and 11 with miscellaneous autoimmune diseases) were tested by the RNase protection assay.…”