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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 Binding of the drug to 23s rRNA is relatively weak ( K d approximately 0.5 pM; M. Stark and E. Cundliffe, unpublished data) but is dramatically enhanced when protein L 11 is also complexed with the RNA.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...

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of ‘domain IV’ of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757‐1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2‐protected oligonucleotides, also derived from domain IV (i.e. from residues 1955‐2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.

The cyanobacterium Aphanocapsa 6714 which grows in the dark on D‐glucose, will take up D‐glucose and the analogue 3‐O‐methyl‐D‐glucose; uptake of each of these compounds was inhibited competitively by the other and by 6‐deoxy‐D‐glucose. This cyanobacterium accumulated 3‐O‐methyl‐D‐glucose up to 100‐fold relative to the medium but did not modify or metabolize it to a significant degree. Intracellular 3‐O‐methyl‐D‐glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D‐glucose and 3‐O‐methyl‐D‐glucose. Although not characterized to the same extent, D‐glucose and 3‐O‐methyl‐D‐glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D‐glucose, was similar. Other cyanobacteria that do not grow on D‐glucose take up this compound at much lower rates which were unaffected by analogues of D‐glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac. It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D‐glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D‐glucose as a growth substrate.

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