The paraoxonase PON1 promoter polymorphism C(-107)T is associated with increased serum glucose concentrations in non-diabetic patients.

AIMS/HYPOTHESIS: Oxidative stress could contribute to diabetes and its complications by predisposing to insulin resistance. Lipid peroxidation products are thought to be one mechanism involved in reduced insulin sensitivity. The serum enzyme, paraoxonase-1, protects lipoprotein lipids from oxidation. We examined the hypothesis that paraoxonase-1 could be associated with abnormal serum glucose concentrations in non-diabetic patients. METHODS: Serum paraoxonase-1 activities and concentrations, as well as paraoxonase-1 gene polymorphisms, were analysed as a function of fasting glucose concentrations in non-diabetic patients and in Type II (non-insulin-dependent) diabetic patients. RESULTS: Serum paraoxonase-1 activities and concentrations were lower (p <0.05) in non-diabetic patients with abnormal fasting glucose concentrations. It was due to a higher frequency of low expressor paraoxonase-1 promoter genotypes in patients with abnormal glucose control. Promoter polymorphisms were independent determinants of abnormal fasting glucose concentrations. Low expressor genotypes were associated with higher glucose concentrations in non-diabetic patients (p = 0.046) and a trend to higher concentrations in Type II diabetic patients. The coding region paraoxonase-1 polymorphisms L55 M and Q192R was not associated with differences in fasting glucose. CONCLUSION/INTERPRETATION. The promoter polymorphism C(-107)T is a marker for abnormal fasting glucose concentrations in non-diabetic patients. It could indicate an active role for paraoxonase-1, possibly pre-disposing to insulin resistance, or linkage of paraoxonase-1 polymorphisms with other gene products implicated in glucose metabolism.

Paraoxonase promoter polymorphism T(-107)C and relative paraoxonase deficiency as determinants of risk of coronary artery disease.

This study tested the hypothesis that promoter polymorphism T(-107)C of the human paraoxonase gene (PON1) is associated with risk of coronary disease. Participants (n=897) were recruited from a cardiology department. All underwent coronary arteriography and were defined as coronary artery disease positive (n=699) or negative (n=198). No association of the promoter genotypes with coronary disease was observed in the overall population, but the high expressor genotype (-107CC) was associated with decreased risk of disease in patients aged 60 years or under in univariate and multivariate analysis independently of established risk factors. A significant deficiency in paraoxonase relative to cholesterol was apparent in patients, even when they were matched with controls for total and low-density lipoprotein cholesterol levels. The -107 polymorphism was not associated with risk in older patients (61 years or over). Age was negatively associated with serum concentrations and activities of paraoxonase; serum paraoxonase was significantly higher in those aged under 61 years than in those aged 61 or over. Age was an independent predictor of paraoxonase concentrations. The results indicate that in this population of patients the promoter polymorphism T(-107)C of the PON1 gene is an independent risk factor for coronary disease in those 60 years or younger. The data are consistent with the hypothesis that lower expression of this anti-oxidant enzyme increases risk of coronary disease. Ageing has also been identified as an independent determinant of serum paraoxonase levels. Ageing is correlated with reduced serum paraoxonase levels, which may compromise the protective influence of enzyme. The results are consistent with the contention that the protective, anti-oxidant capacity of high density lipoproteins is at least in part genetically determined.

Decreased stability of the M54 isoform of paraoxonase as a contributory factor to variations in human serum paraoxonase concentrations.

There are considerable variations in serum concentrations of the high density lipoprotein (HDL)-associated enzyme, paraoxonase (PON), which is an important determinant of the antioxidant capacity of HDL. The present study examined the hypothesis that differences in the stability of isoforms arising from the coding region L54M polymorphism could contribute to such variations. A model system was developed using transfected Chinese hamster ovary cells to secrete recombinant PON corresponding to human L or M isoforms. The recombinant peptides exhibited the molecular properties of human serum PON. They formed complexes with lipoproteins in culture medium, notably binding to apolipoprotein A-I-containing particles. The enzymatic properties of the recombinant isoforms were comparable to those of human serum PON. The recombinant M isoform lost activity more rapidly and to a greater extent than the recombinant L isoform [26.0 +/- 3.0% vs. 14.0 +/- 1.0% (phenylacetate substrate) and 36.1 +/- 2.0% vs. 19.3 +/- 2.0% (paraoxon substrate) over 96 h (P < 0.01)] in medium containing fetal calf serum or PON-free human serum. Addition of a protease inhibitor resulted in retention of activity by both isoforms. Parallel results were obtained in incubation studies of human serum from donors homozygous LL or MM for the L54M polymorphism. Enzyme activity was lost more rapidly and to a greater extent from MM than LL sera (P < 0.01). A parallel loss of PON peptide mass was also observed, with a significantly greater loss from MM homozygotes (P < 0.001). It corresponded to the appearance of a smaller molecular mass band on SDS-PAGE analysis. Direct analysis of the proteolytic effect using HDL isolated from homozygotes and incubated with purified kallikrein confirmed the greater loss of activity from MM homozygotes and the protective effect of proteolysis inhibitor. The results provide evidence for lesser stability of the M54 isoform of PON, apparently involving greater susceptibility to proteolysis. It provides one mechanism to explain variations in serum levels of PON and has implications for the antioxidant capacity of HDL.

Serum paraoxonase is reduced in type 1 diabetic patients compared to non-diabetic, first degree relatives; influence on the ability of HDL to protect LDL from oxidation.

Paraoxonase is a serum enzyme with an anti-oxidant function, protecting low density lipoproteins (LDL) from oxidative modifications. Diabetic patients are suggested to be at greater risk of oxidative stress, which may contribute to the significantly higher incidence of vascular disease in this population. Less efficient protection mechanisms may be one feature of the greater susceptibility to oxidation in diabetes. In this context, the present study examined the hypothesis that serum paraoxonase is reduced in type 1 (insulin-dependent) diabetic patients and that the reduction can affect the anti-oxidant capacity of HDL. Serum paraoxonase concentrations and activities were compared in type 1 patients and first degree, non-diabetic relatives with particular attention paid to the confounding effects of paraoxonase gene polymorphisms. In addition, the ability of HDL-paraoxonase to protect low density lipoproteins from oxidation was analysed in an in vitro system. Serum concentrations and enzyme activities of paraoxonase were significantly lower in type 1 patients compared to non-diabetic, first degree relatives. The differences were independent of promoter and coding region polymorphisms, which influence serum concentrations and activities of the enzyme. Overall, paraoxonase concentrations were a mean 13.3+/-4.5% lower (P<0.02) in type 1 patients. Specific activities did not differ between diabetic and non-diabetic groups. The concentration ratios of LDL cholesterol:paraoxonase (1.37+/-0.51 vs. 1.18+/-0.37, P=0.003) and apolipoprotein B:paraoxonase (0.84+/-0.33 vs. 0.71+/-0.40; P=0.012) were significantly higher in diabetic patients, consistent with a reduced capacity to protect LDL from oxidation. In vitro oxidation studies showed that a significantly higher level of lipid hydroperoxides was generated in LDL in the presence of HDL, containing paraoxonase levels equivalent to those of type 1 patients, compared to HDL containing paraoxonase levels equivalent to those of control subjects (mean difference 8.1%, P<0.05). The study demonstrates that serum concentrations of the antioxidant enzyme paraoxonase are significantly lower in type 1 (insulin-dependent) diabetic patients compared to non-diabetic, first-degree relatives, independently of known gene polymorphisms. Concentrations are reduced to an extent that can affect its anti-oxidant capacity. The results are consistent with the contention that modifications to serum paraoxonase in type 1 patients can increase risk of lipoprotein oxidation and, consequently, risk of vascular disease.

Promoter polymorphism T(-107)C of the paraoxonase PON1 gene is a risk factor for coronary heart disease in type 2 diabetic patients.

The serum enzyme paraoxonase (PON) protects LDLs from oxidative stress. We recently identified promoter polymorphisms of the PON gene that strongly affect gene expression and serum levels of the enzyme. The present study tested the hypothesis that promoter polymorphism T(-107)C could be a risk factor for vascular disease in type 2 diabetic patients by virtue of its ability to modulate serum concentrations of the antioxidant enzyme. The low-expressor genotype (TT) was associated with significantly lower serum PON concentrations, and it was over-represented in type 2 diabetic patients with coronary heart disease (CHD) (TT vs. TC+CC: odds ratio [OR] 1.64 [95% CI 1.03-2.61], P < 0.05). The association of the low-expressor genotype with an increased risk of disease was independent of other risk factors, including the coding region Q191R polymorphism (OR 2.12 [95% CI 1.19-3.70], P = 0.01). However, an interaction of the promoter polymorphism with the Q191R polymorphism, which was previously identified as an independent risk factor, was observed. The low-expressor promoter allele (-107T) associated with the high-risk 191R allele showed a lower-than-expected level of risk (OR 2.21 vs. the expected 4.76). The data are consistent with the hypothesis that low expression of the antioxidant enzyme PON increases the risk of CHD. Moreover, the promoter polymorphism appears to have a modulating effect on risk that is associated with the coding region polymorphism Q191R. This study indicates a strong genetic component to the antioxidant capacity of HDLs.

Smoking is associated with reduced serum paraoxonase activity and concentration in patients with coronary artery disease.

BACKGROUND: Paraoxonase is an HDL-associated enzyme that protects lipoproteins from oxidative modifications. Smoking is a major cardiovascular risk factor that promotes lipid peroxidation. Cigarette smoke has been shown in vitro to inhibit paraoxonase. The present study examined the hypothesis that smoking is associated with modulated serum activities and concentrations of paraoxonase. METHODS AND RESULTS: Coronary artery disease was assessed with the use of coronary arteriography in participants recruited from a hospital cardiology division. Medical and lifestyle data were obtained, and a fasting blood sample was provided. Three smoking categories were established (never, ex-smokers, and current smokers), and serum paraoxonase variables were compared among them. The activities and concentrations of paraoxonase were significantly lower in current than in never smokers. Ex-smokers had values comparable to those of never smokers. Ex-smokers who had recently stopped (<3 months) had activities and concentrations comparable to those of current smokers; values returned to the levels of never smokers within 2 years of cessation of smoking. Smoking status was an independent determinant of paraoxonase activity and concentration in multivariate analysis. Finally, lower paraoxonase was associated with more severe coronary disease and a reduced capacity to protect LDL from oxidation. CONCLUSIONS: Smoking is independently associated with significant decreases in serum paraoxonase activities and concentrations, which normalize within a relatively short time of cessation. Lower serum paraoxonase is linked to more severe coronary artery disease and a lower antioxidant capacity. The data are consistent with the hypothesis that smoking modifies serum paraoxonase such that there is an increased risk of coronary artery disease due to a diminished capacity to protect lipoproteins from oxidative stress.

Promoter polymorphisms of human paraoxonase PON1 gene and serum paraoxonase activities and concentrations.

Paraoxonase (PON) is a serum enzyme with a wide species distribution. It protects lipoproteins from toxic oxidative modifications and is an antiatherogenic mechanism of major potential. Activity levels of PON are major determinants of the protective function; consequently, factors that influence PON levels are of particular relevance. The present study has identified 3 polymorphisms in the promoter region of the human PON1 gene. Cell transfection studies have revealed their variable impact on promoter activity, with up to 2-fold differences in reporter gene expression. Genotyping studies have established that the polymorphisms are frequent in the population, a finding that is consistent with a major impact on PON concentrations. The physiological relevance of the polymorphisms was underlined by showing that they are associated with highly significant differences in serum concentrations and activities of PON. The study thus firmly establishes a genetic basis for variations in serum PON levels and, consequently, serum PON activity. It is consistent with the suggestion that variations in a major antioxidant function of high density lipoprotein are, to an important degree, genetically determined.

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre.

A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.

Two alleles of the human paraoxonase gene produce different amounts of mRNA. An explanation for differences in serum concentrations of paraoxonase associated with the (Leu-Met54) polymorphism.

In a recent study we demonstrated that a polymorphism of the human paraoxonase gene affecting position 54 was linked to variations in serum concentrations of the enzyme. L allele carriers (leucine at position 54) have significantly higher concentrations of paraoxonase than M allele carriers (methionine at position 54). In the present study we examined the hypothesis that differences in mRNA production could contribute to variations in serum concentrations. Relative concentrations of L and M type mRNA were analyzed in total RNA extracted from heterozygous liver samples. This was achieved by cDNA synthesis, polymerase chain reaction amplification of the cDNA fragment containing the 54 polymorphism and restriction analysis to identify radiolabeled end fragments of L and M alleles. An allele mixing experiment using total RNA from liver samples of LL and MM homozygotes demonstrated the sensitivity of the approach to changes in the relative concentrations of each type of RNA. In 8 of 10 heterozygous samples, an excess of L allele type mRNA was observed. Overall there was a significantly higher level of L type mRNA (L:M ratio of 2.51 +/- 1.41, n = 10, P < .01). These results support our hypothesis that increased concentrations of serum paraoxonase arise from greater production of L allele mRNA. In two samples, the L:M ratio was close to or below 1.0. This is consistent with the known spectrum of paraoxonase serum concentrations associated with the L and M alleles and suggests that factor(s) that preferentially modulate allele expression are usually, but not uniformly, associated with the L allele.

Molecular cloning of a preprohormone from Hydra magnipapillata containing multiple copies of Hydra-L Wamide (Leu-Trp-NH2) neuropeptides: evidence for processing at Ser and Asn residues.

The simple, freshwater polyp Hydra is often used as a model to study development in cnidarians. Recently, a neuropeptide, < Glu-Gln-Pro-Gly-Leu-Trp-NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a polyp. Here, we have cloned a preprohormone from Hydra magnipapillata containing 11 (eight different) immature neuropeptide sequences that are structurally related to the metamorphosis-inducing neuropeptide from sea anermones. During the final phase of our cloning experiments, another research team independently isolated and sequenced five of the neuropeptides originally found on the preprohormone. Comparison of these mature neuropeptide structures with the immature neuropeptide sequences on the preprohormone shows that most immature neuropeptide sequences are preceded by Ser or Asn residues, indicating that these residues must be novel processing sites. Thus, the structure of the Hydra preprohormone confirms our earlier findings that cnidarian preprohormones contain unusual or novel processing sites. Nearly all neuropeptide copies located on the Hydra preprohormone will give rise to mature neuropeptides with a C-terminal Gly-Leu-Trp-NH2 sequence (the most frequent one being Gly-Pro-Pro-Pro-Gly-Leu-Trp-NH2; Hydra-LWamide l; three copies). Based on their structural similarities with the metamorphosis-inducing neuropeptide from sea anemones, the mature peptides derived from the Hydra-LWamide preprohormone are potential candidates for being developmentally active neurohormones in Hydra.

General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid.

Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation procedure is described for both organisms. The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S. acidocaldarius. A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S. acidocaldarius. Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection. Growth inhibition studies indicate that both P. furiosus and S. acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol. Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S. acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P. furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA). These mutated genes are being developed as selective markers. Moreover, two beta-galactosidase genes from P. furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95 degrees C with O-nitrophenyl beta-D-galactopyranoside as substrate.

Peptides in the nervous systems of cnidarians: structure, function, and biosynthesis.

Cnidarians are the lowest animal group having a nervous system and it was probably within this phylum or in a related ancestor group that nervous systems first evolved. The primitive nervous systems of cnidarians are strongly peptidergic. From a single sea anemone species, Anthopleura elegantissima, 17 different neuropeptides have been isolated so far, and we expect that many more neuropeptides (more than 30) must be present. All peptides are localized in neurons of cnidarians and we have demonstrated the presence of some of the peptides in neurosecretory dense-cored vesicles. Most neuropeptides have an excitatory or inhibitory action on whole cnidarians, muscle preparations, and isolated muscle cells, suggesting that these peptides are neurotransmitters or neuromodulators. One neuropeptide induces metamorphosis in planula larvae to become a polyp. This shows that cnidarian neuropeptides also are involved in developmental processes, such as cell differentiation and pattern formation. We have cloned the preprohormones for most of the cnidarian neuropeptides. These preprohormones have a high copy number of the immature neuropeptide sequence, which can be up to 37 neuropeptide copies per precursor molecule. In addition to well-known, "classical" processing enzymes, novel prohormone processing enzymes must be present in cnidarian neurons. These include a processing enzyme hydrolyzing at the C-terminal sides of acidic (Asp and Glu) residues and a dipeptidyl aminopeptidase digesting at the C-terminal sides of N-terminally located X-Pro and X-Ala sequences. All this shows that the primitive nervous systems of cnidarians are already quite complex, and that neuropeptides play a central role in the physiology of these animals.

Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis-inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing.

Neuropeptides are an important group of hormones mediating or modulating neuronal communication. Neuropeptides are especially abundant in evolutionarily "old" nervous systems, such as those of cnidarians, the lowest animal group having a nervous system. Cnidarians often have a life cycle including a polyp, a medusa, and a planula larva stage. Recently, a neuropeptide, < Glu-Gln-Pro-Gly-Leu-Trp-NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a hydropolyp [Leitz, T., Morand, K. & Mann, M. (1994) Dev. Biol. 163, 440-446]. Here, we have cloned the precursor protein for this metamorphosis-inducing neuropeptide from sea anemones. The precursor protein is 514-amino acid residues long and contains 10 copies of the immature, authentic neuropeptide (Gln-Gln-Pro-Gly-Leu-Trp-Gly). All neuropeptide copies are preceded by Xaa-Pro or Xaa-Ala sequences, suggesting a role for dipeptidyl aminopeptidase in neuropeptide precursor processing. In addition to these neuropeptide copies, there are 14 copies of another, closely related neuropeptide sequence (Gln-Asn-Pro-Gly-Leu-Trp-Gly). These copies are flanked by basic cleavage sites and, therefore, are likely to be released from the precursor protein. Furthermore, there are 13 other, related neuropeptide sequences having only small sequence variations (the most frequent sequence: Gln-Pro-Gly-Leu-Trp-Gly, eight copies). These variants are preceded by Lys-Arg, Xaa-Ala, or Xaa-Pro sequences, and are followed by basic cleavage sites, and therefore, are also likely to be produced from the precursor. Thus, there are at least 37 closely related neuropeptides localized on the precursor protein, making this precursor one of the most productive preprohormones known so far. This report also shows that unusual processing sites are common in cnidarian preprohormones.

Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity.

The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movement of the 5' end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3' ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P/E sites on peptide bond formation.

Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre.

The function of the highly conserved and accessible region of domain IV of 23S rRNA (positions 1900-1981 in Escherichia coli 23S rRNA) was investigated by subjecting it to a random mutagenesis procedure that produced single-site mutations efficiently. Nine single-site mutants were selected that were recessive lethal. High levels of mutated 23S rRNA were expressed in E. coli and extracted ribosomes were investigated for their content of mutated rRNA. The peptidyl transferase activity of the ribosomes was also estimated using a newly developed method involving selective inhibition of chromosome-encoded ribosomes by clindamycin. Two of the mutants, U1940A and U1955G, yielded 50S subunits that were defective in subunit-subunit association but active in peptidyl transferase activity and five, U1926C, U1946C, U1979C, U1982A and G1984A, produced 50S subunits that were defective in both subunit-subunit interactions and peptidyl transferase activity. We infer that the large conserved rRNA region generates a complex structure that plays an essential role in maintaining and modulating subunit-subunit interactions and argue that its involvement in the peptidyl transferase centre is secondary, possibly involving the correct alignment of protein L2.

Cross-hypersensitivity effects of mutations in 23 S rRNA yield insight into aminoacyl-tRNA binding.

Halobacterium halobium cells contain one set of rRNA genes per genome. They have been used to characterize spontaneous mutants, carrying single nucleotide mutations in their rRNAs, that are resistant to different ribosomal antibiotics. Here we demonstrate that two different mutants that show resistance to thiostrepton, an inhibitor of the ribosomal GTPase-centre, are hypersensitive to amicetin, and other antibiotics, which act at the peptidyl transferase centre. Conversely, an amicetin-resistant mutant exhibits hypersensitivity to thiostrepton. A model is presented in which the two mutated sites, which are widely separated in the primary and secondary structure of 23 S rRNA, both participate in A-site binding of aminoacyl-tRNA.

A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome.