A previous survey of upstream sequences of tRNA genes from the archaebacterium Methanococcus vannielii has revealed that there are two boxes of sequence homology: A box "A" of about 20 conserved nucleotides at a distance of 30 to 49 basepairs upstream from the gene and a box "B" 18 to 19 nucleotides downstream from box "A" (Wich, G., Sibold, L., and Bock, A. (1985) System. Appl. Microbiol. (in press). Nuclease Si mapping experiments were carried out with two of these tRNA transcriptional units and with a ribosomal RNA operon, to determine whether these consensus sequences have a function in the initiation of transcription. Use was made of the fact that cells from Methanococcus accumulate primary transcript and processing intermediates of ribosomal RNA under conditions of protein synthesis inhibition. The following results were obtained: (i) Transcription in all three systems starts at the G within the conserved trinucleotide TGC of box "B". Since the box "B" motif, 5'TGCaagT3', also occurs at the site of transcription initiation of protein encoding genes, both in methanogenic and halophilic organisms, it appears to constitute a frequently used transcription start signal within these archaebacterial groups. (ii) The box "A" motif occurs with constant spacing, relative to box "B", in all 10 tRNtA and ribosomal RNA transcriptional units investigated from Methanococcus. Since it is not present in the leader region of genes coding for proteins, it seems to function as a specific element which is required for the expression of genes for stable RNA. (iii) Termination of transcription of the ribosomal RNA operon from Methanococcus occurs at a distinct T within an oligo-T stretch immediately downstream from the 3'-terminal 5S RNA gene. This signal occurs in all 3'-flanking regions of transcriptional units for stable RNA from the Methanococcus strains studied. Termination signals for stable RNA genes in Methanococcus appear to be similar with those of stable RNA genes in eukaryotes. (iv) By nuclease Sl mapping a recognition site was identified for a processing enzyme involved in the maturation of preribosomal RNA.

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...

Halobacterium (H.) halobium and H. cutirubrum mutants resistant to the anti-80S ribosome targeted inhibitor anisomycin were isolated. Three classes of mutants were obtained: Class I displayed a minimal inhibitory concentration (MIC) to anisomycin of 10 micrograms/ml, class II of 25 micrograms/ml and class III of at least 400 micrograms/ml. In vitro polyphenylalanine synthesis assays demonstrated that in those cases tested resistance was a property of the large ribosomal subunit. By primer extension analysis, each mutation class could be correlated with a distinct base change within the peptidyltransferase loop of 235 rRNA. In class I A2472 was changed to C, in class II G2466 was changed to C and in the high-level resistant class III C2471 was replaced by U. A. double mutant - obtained by selection of a class I mutant for high-level anisomycin resistance - acquired the C2471 to U replacement of class III in addition to the class I mutation. The results provide information on the action of a eukaryotic protein synthesis inhibitor on archaebacterial ribosomes and demonstrate the suitability of organisms with a single rRNA transcriptional unit on the chromosome for direct selection of mutations in ribosomal RNA.

Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of stearate (18:0) to oleate (18:1n-9) and of palmitate (16:0) to palmitoleate (16:1), which are key steps in triglyceride synthesis in the fatty acid metabolic network. This study investigated the role of SCD1 in fatty acid metabolism in HepG2 cells using SCD1 inhibitors and stable isotope tracers. HepG2 cells were cultured with [U-13 C]stearate, [U-13 C]palmitate, or [1,2-13 C]acetate and (1) DMSO, (2) compound CGX0168 or CGX0290, or (3) trans-10,cis-12 conjugated linoleic acid (CLA).13 C incorporation into fatty acids was determined by GC-MS and desaturation indices calculated from the respective ion chromatograms. FAS, SCD1, peroxisome proliferator-activated receptor a, and peroxisome proliferator-activated receptor g mRNA levels were assessed by semiquantitative RT-PCR. The addition of CGX0168 and CGX0290 decreased the stearate and palmitate desaturation indices in HepG2 cells. CLA led to a decrease in the desaturation of stearate only, but not palmitate. Comparison of desaturation indices based on isotope enrichment ratios differed, depending on the origin of saturated fatty acid. SCD1 gene expression was not affected in any group. In conclusion, the differential effects of SCD1 inhibitors and CLA on SCD1 activity combined with the dependence of desaturation indices on the source of saturated fatty acid strongly support the compartmentalization of desaturation systems. The effects of SCD1 inhibition on fatty acid composition in HepG2 cells occurred through changes in the dynamics of the fatty acid metabolic network and not through transcriptional regulatory mechanisms. The enzyme stearoyl-coenzyme A desaturase (SCD) is important in the conversion of saturated fatty acids to monounsaturated fatty acids (MUFAs). The isoform SCD1 catalyzes the desaturation of palmitate (16:0) to palmitoleate (16:1n-9) and of stearate (18:0) to oleate (18:1n-9). Palmitoleate and oleate are the main MUFAs that constitute membrane phospholipids, triglycerides, wax esters, and cholesteryl esters. Because an inappropriate ratio of MUFA to saturated fatty acid can affect membrane lipid fluidity and lipoprotein metabolism, the effects of SCD have been implicated not only in obesity but also in diabetes, atherosclerosis, and cancer (1-5).Mouse models of SCD1 deficiency have demonstrated effects on body weight and lipid metabolism. Asebia mice have a naturally occurring homozygous mutation in SCD1 (6). These mice lack sebaceous glands and have alopecia and dry skin. In addition, they are lean and have impaired hepatic ability to synthesize cholesteryl esters and triglycerides (7). Ntambi and colleagues (8) created a knockout mouse for SCD1. These mice have decreased adiposity, increased insulin sensitivity, and are resistant to diet-induced weight gain. These properties of the SCD1 2/2 mouse have generated much interest in SCD as a potential target for obesity prevention in humans.In the mouse and rat, inhibition of SCD1 using antisense oligonucleotides has been...

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