The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA.

The peptidyl transfer site has been localized at the centre of domain V of 23S-like ribosomal RNA (rRNA) primarily on the basis of a chloramphenicol binding site. The implicated region constitutes an unstructured circle in the current secondary structural model which contains several universally conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed in vivo on a plasmid-encoded rRNA (rrnB) operon and each one yielded dramatically altered phenotypes. Cells exhibiting A2060----C or A2450----C transversions were inviable and it was shown by inserting the mutated genes after a temperature-inducible promoter that the mutant RNAs were directly responsible. In addition, a G2502----A transition caused a decreased growth rate, probably due to a partial selection against mutant ribosome incorporation into polysomes, while an A2503----C transversion produced a decreased growth rate and conferred resistance to chloramphenicol. All of the mutant RNAs were incorporated into 50S subunits, but while the two lethal mutant RNAs were strongly selected against in 70S ribosomes, the plasmid-encoded A2503----C RNA was preferred over the chromosome-encoded RNA, contrary to current regulatory theories. The results establish the critical structural and functional importance of highly conserved nucleotides in the chloramphenicol binding region. A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface.     

Mutants with base changes at the 3'-end of the 16S RNA from Escherichia coli. Construction, expression and functional analysis

The functionally important 3' domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid-encoded 16S RNA gene. By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U. Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the lambda PL promoter. In a strain coding for the temperature-sensitive lambda repressor cI857 the mutant RNAs could be expressed conditionally. We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes. However, all three mutants inhibited the formation of stable 70S particles to various degrees. The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis. While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent. Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo. The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes. The results underline the importance of the highly conserved nucleotides in the 3' domain of the 16S RNA for ribosomal function.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

Nucleotide sequence and structure of cytoplasmic 5S RNA and 5.8S RNA of Chlamydomonas reinhardii.

Three small RNAs of the cytoplasmic 8OS ribosomes of the green unicellular alga Chlamydomonas reinhardii have been sequenced. They include two species of ribosomal 5S RNA, a major and a minor one of 122 and 121 nucleotides respectively, which differ from each other by 17 bases, and also the ribosomal 5.8S RNA of 156 nucleotides. Novel structural features can be recognized in the 5S RNAs of C. reinhardii by a comparison with published 5S RNA sequences. In addition the secondary structure of these small RNA molecules has been examined using a newly developed method based on differential nuclease susceptibility.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.     

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.     

Structure-function relationship of Rous sarcoma virus leader RNA.

Cells infected by RSV synthesize viral 35S RNA as well as subgenomic 28S and 22S RNAs coding for the Env and Src genes respectively. In addition, at least the 5' 101 nucleotides of the leader are also conserved and we have shown previously that this sequence contains a strong ribosome binding site (J.-L. Darlix et al., J. Virol. 29, 597). We now report the RNA sequence of Rous Sarcoma virus (RSV) leader RNA and propose a folding of this 5' untranslated region which brings the Cap, the initiation codon for Gag and the strong ribosome binding site close to each other. We also show that ribosomes protect a sequence just upstream from initiator Aug of Gag in vitro, and believed to interact with part of the strong ribosome binding site according to the folding proposed for the leader RNA.     

The mechanism of action of trichosanthin on eukaryotic ribosomes--RNA N-glycosidase activity of the cytotoxin.

Trichosanthin is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the mechanism of action of trichosanthin on eukaryotic ribosomes was studied. A fragment of about 450 nucleotides was released from 28S ribosomal RNA after treatment of rat liver ribosome with trichosanthin and its isolated ribosomal RNAs were treated with aniline. Analysis of nucleotide sequence of 5' terminus of this fragment revealed that the aniline-sensitive site of the phosphodiester bond was between positions A4324 and G4325 in the 28S rRNA. Adenine was recovered by ion-exchange column chromatography from the 50% ethanol soluble fraction of the reaction mixture in which rat liver ribosomes were treated with trichosanthin. Thin-layer chromatographic analysis indicated that 1 mol of adenine was released from 1 mol of ribosomes. When the ribosomes were incubated with trichosanthin in the presence of inorganic [32P]phosphate, little incorporation of radioactivity into 28S rRNA was observed, indicating that the release of adenine was not mediated by phosphorolysis. These results demonstrate that trichosanthin inactivates the ribosomes by cleaving the N-C glycosidic bond of adenylic acid at 4324 of 28S rRNA in a hydrolytic fashion.     

Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA

TYMV RNA supports the translation of two proteins, p69 and p206, from AUG initiation codons 7 nucleotides apart. We have studied the translation of this overlapping dicistronic mRNA with luciferase reporter RNAs electroporated into cowpea protoplasts and in toe-printing studies that map ribosomes stalled during initiation in wheat germ extracts. Agreement between these two assays indicates that the observed effects reflect ribosome initiation events. The robust expression from the downstream AUG206 codon was dependent on its closeness to the upstream AUG69 codon. Stepwise separation of these codons resulted in a gradual increase in upstream initiation and decrease in downstream initiation, and expression was converted from dicistronic to monocistronic. Selection by ribosomes for initiation between the nearby AUG codons was responsive to the sequence contexts that govern leaky scanning, but the normally strong position effect favoring upstream initiation was greatly diminished. Similar dicistronic expression was supported for RNAs with altered initiation sequences and for RNAs devoid of flanking viral sequences. Closely spaced AUG codons may thus represent an under-recognized strategy for bicistronic expression from eukaryotic mRNAs. The initiation behavior observed in these studies suggests that 5'-3' ribosome scanning involves backward excursions averaging about 15 nucleotides.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii.

The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients. The sedimentagion coefficient of the principal peak was 66-70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C. reinhardtii, and other organisms. The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit. When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S. Thus the 69 S peak was an artifact caused by dissociation during centrifugation. Peaks that contained chloroplast ribosomal RNAs were also observed at '60 S' and '45 S' when chloroplast ribosomes were centrifuged unfixed at high velocity. The amounts of '60 S' and '45 S' components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged. The '60 S', and '45 S' components were identified as large, and small subunits of chloroplast ribosomes, respectively. The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli. Similar explanations appear to apply to both. We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of 'tight' couples, and incomplete separation of subunits. Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of 'loose' couples.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.