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PubMed1987

Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site.

We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.

Base Sequence↗
PubMed1986

Analysis of the RNA structural elements involved in the binding of the transcription factor III A from Xenopus laevis.

Xenopus laevis 5S rRNA isolated from 7S particles or transcribed in vitro is found to adopt two alternative conformations. These two conformers contain different structural elements within the major TF III A binding domain, which, when isolated as RNA fragments, still interact with the transcription factor. Chemical modification of easily accessible adenines in their N-1 position does not have any measurable effect on the binding of 5S rRNA to TF III A. These observations are in support of the idea that only a small amount of conserved sequence information is required for the binding of the transcription factor, whereas specific secondary structure features seem to be essential.

Animals↗
PubMed1978

Studies on the conformation of the 3' terminus of 18-S rRNA.

We have studied the conformation of the 3' end of 18-S RNA from human, hamster and Xenopus laevis cells. The 3'-terminal oligonucleotide in a T1 ribonuclease digest of 18-S RNA from HeLa cells was identified, using a standard fingerprinting method. The sequence (G)-A-U-C-A-U-U-A, established by Eladari and Galibert for HeLa 18-S rRNA, was confirmed. An identical 3' terminus is present in hamster fibroblasts and Xenopus laevis cells. The ease of identification of this oligonucleotide has enabled us to quantify its molar yield relative to several other oligonucleotides, and hence to analyse the 3' terminus by several conformation probes. Its sensitivity to S1 nuclease, limited T1 ribonuclease digestion, bisulphite modification and carbodiimide modification was consistent with the terminal oligonucleotide being in a highly exposed conformation. The m6/2A-m6/2A-C-containing sequence of 18-S rRNA also appears to be in an exposed location on the basis of three of these probes.

Animals↗
PubMed2002

A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis.

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

Animals↗
PubMed1997

Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34(cdc2 )phosphorylation site in the consensus S50PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34(cdc2 )from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34(cdc2 )in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13(suc1)-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34(cdc2(kinase. In vivo 32P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34(cdc2 )kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA.

Alanine↗
PubMed1995

Pseudoknot in domain II of 23 S rRNA is essential for ribosome function.

The structure of domain II in all 23 S (and 23 S-like) rRNAs is constrained by a pseudoknot formed between nucleotides 1005 and 1138, and between 1006 and 1137 (Escherichia coli numbering). These nucleotides are exclusively conserved as 1005C.1138G and 1006C.1137G pairs in all Bacteria, Archaea and chloroplasts, whereas 1005G.1138C and 1006U.1137A pairs occur in Eukarya. We have mutagenized nucleotides 1005C-->G, 1006C-->U, 1137G-->A and 1138G-->C, both individually and in combinations, in a 23 S rRNA gene from the bacterium E. coli. The ability of 23 S rRNA to support cell growth is reduced when either of these base-pairs is disrupted, and it is completely abolished upon disruption of both base-pairs. Each mutant 23 S rRNA is assembled into 50 S subunits, but the mutant subunits do not stably interact with 30 S to engage in protein synthesis. Enzymatic and chemical probing of ribosomal particles reveals increased accessibility in the rRNA structure close to the sites of the mutations. The degree to which the mutations increase rRNA accessibility correlates with the severity of their phenotypic effects. Nucleotide 1131G is extremely reactive to dimethyl sulphate modification in wild-type subunits and ribosomes, but is rendered unreactive when either the pseudoknot is broken or when the r-proteins are removed. The structure of the pseudoknot region is possibly influenced by interaction of an r-protein at or close to the pseudoknot. Re-establishing the pseudoknot Watson-Crick interactions with one "eukaryal" (1005G.1138C or 1006U.1137A) pair and one "bacterial" C.G pair largely restores the structure and function of the rRNA. Bacterial ribosomes containing both these eukaryal pairs also participate in protein synthesis, although at much reduced efficiency, and the structure of their pseudoknot region is partially open and accessible.

Base Sequence↗
PubMed1984

Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate.

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound ("engaged") and unbound ("free") forms revealed that the steroid-mediated inhibition of the activity of the "engaged" form of the enzyme was not accompanied by significant changes in "free" pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.

Animals↗
PubMed1998

SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction.

Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs. Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells. We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide. However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex. Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.

Animals↗
PubMed2004

Npa1p, a component of very early pre-60S ribosomal particles, associates with a subset of small nucleolar RNPs required for peptidyl transferase center modification.

We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.

Amino Acid Sequence↗
PubMed2006

Identification of Escherichia coli m2G methyltransferases: I. the ycbY gene encodes a methyltransferase specific for G2445 of the 23 S rRNA.

N2-methylguanosine 2445 of the 23 S rRNA is located in a cluster of modified nucleotides concentrated at the peptidyl transferase center of the ribosome. Here we describe the identification of a gene, ycbY, as encoding an enzyme responsible for methylation of G2445. Knock-out of the ycbY gene leads to loss of modification at G2445 as revealed by reverse transcription. The modification is restored in the ycbY knock-out strain if co-transformed with a plasmid expressing the ycbY gene product. Recombinant YcbY protein is able to methylate 23 S rRNA purified from the ycbY knock-out strain in vitro, assembled 50 S subunits are not a substrate for the methylase. Knock-out of the ycbY gene leads to growth retardation. Growth competition with the parental wild-type strain leads to a gradual decrease in the knock-out strain cells proportion in the media. It is likely that the G2445 modification is necessary for prevention of non-functional secondary or tertiary structure formation at the peptidyl transferase center. Based on these results we suggest that YcbY be renamed to RlmL in accordance with the accepted nomenclature for rRNA methyltransferases.

Base Sequence↗
PubMed1977

Hypermodified alkali-stable dinucleotide sequences in each of the high-molecular-weight (26S and 18S) ribosomal RNA species of wheat.

Two hypermodified, alkali-stable dinucleotide sequences, each containing a base modification in addition to sugar methylation, are known to be present in wheat embryo 26S + 18S rRNA (Gray, M.W. (1974) Biochemistry 13, 5453-5463). Quantitative analysis of unfractionated 26S + 18S rRNA had suggested that each of these sequences (Cm-psi p and psi m-Ap, where Cm=O2'-methylcytidine and psi m-O2'-methylpseudouridine) was present in either the 18S or the 26S rRNA species, but not the both, at a frequency of not more than once per chain. In the study reported here, the individual 32P-labeled 18S and 26S rRNA species were isolated from viable wheat embryos germinated in the presence of [32P]orthophosphate. From analyses of phosphodiesterase and alkaline hydrolysates of the separated [32P]RNAs, we conclude that psi m-Ap is confined to wheat cytosol 18S rRNA, whereas Cm-psi p is localized in wheat cytosol 26S rRNA. The presence of psi m in the 18S rRNA of wheat stands in contrast with the situation in animal cells, where this hypermodified nucleoside is located in the 28S rRNA (Khan, M.S.N. & Maden, B.E.H. (1976) J. Mol. Biol. 101, 235-254).

Cytosol↗
PubMed1999

Molecular mechanisms of resistance to commonly used non-betalactam drugs in Streptococcus pneumoniae.

This article reviews the molecular mechanisms of resistance to fluoroquinolones, erythromycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole in Streptococcus pneumoniae. Resistance to fluoroquinolones primarily involves mutations in the DNA gyrase gene, gyrA, and in the topoisomerase IV genes, parC and parE, although in vitro studies have indicated that some strains may use an efflux mechanism for resistance to certain fluoroquinolones. Ciprofloxacin resistance results from initial and necessary mutations in ParC leading to low-level resistance and subsequent mutations in GyrA leading to high-level resistance. Sparfloxacin resistance results from initial mutations in GyrA, with ParC mutations occurring subsequently. A single amino acid substitution in ParE has also been associated with low-level resistance in S pneumoniae. Two mechanisms have been described for resistance to erythromycin. Coresistance to macrolides, lincosamides, and streptogramin B type antibiotics is a result of modification of the ribosome through methylation of an adenine residue in domain V of the 23S rRNA. This methylation is encoded by the methylase gene, ermAM. Resistance only to 14-and 15-membered macrolides is a result of efflux of the antibiotic from the cell, encoded by the gene, mefE, in S pneumoniae, and appears to be rapidly emerging as the predominant mechanism of resistance to erythromycin in many countries. The production of chloramphenicol acetyltransferase, an enzyme capable of catalyzing the conversion of chloramphenicol to its nonfunctional 1-acetoxy, 3-acetoxy, and 1,3-diacetoxy derivatives, leads to chloramphenicol resistance in S pneumoniae. Chloramphenicol acetyltransferase is encoded by a cat gene identical to the cat gene from the Staphylococcus aureus plasmid, pC194. Tetracycline resistance occurs through ribosomal protection encoded by the genes tet(M) and tet(O). It is possible that the Tet(M) and Tet(O) proteins cause tetracycline to be released from the ribosome, although the precise mechanism remains unclear. Resistance to trimethoprim is mediated through a single amino acid substitution in the chromosomal dihydrofolate reductase gene of S pneumoniae, which is thought to disrupt the bond with trimethoprim without affecting the action of the dihydrofolate reductase. Sulphonamide resistance appears to result from repetitions of one or two amino acids in the chromosomal dihydropteroate synthase. Although resistance exists to nearly all antimicrobial agents used in the treatment of S pneumoniae infections, ongoing research into new or alternative therapies is encouraging.

Animals↗
PubMed1977

The origin of the protein synthesis mechanism.

The origin and development of the protein synthesis mechanism is considered in four successive steps. The genetic code is supposed to be controlled by the relative amount (availability) of various amino acids and nucleotides on the one hand, and utility on each amino acid in the polypeptide. on the other hand. Thus, more simple (inutile) and abundant amino acids tended to correspond to codons which were rich in the less frequent base species, G and C. Features of primitive tRNA in the discrimination of amino acid are discussed. Primitive tRNA is proposed to have a discriminator site for amino acid and, separated from it, an anticodon site for interaction with nucleotides. A hypothetical course of subdivision of various nucleic acid species is proposed. In the scheme, mRNA and ribosomal RNA (rRNA) were derived from more primitive insoluble RNA. DNA appeared in the late, not first, step of the development. Several other aspects of evolutionary development of the whole protein synthesis mechanism, e.g., role of the discriminator site on primitive tRNA, modification and subdivision of code catalogue into a more precise specification of amino acids, and possible primordial interactions between tRNA and tRNA-binding sites on insoluble rRNA, are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Role of the spacer boxA of Escherichia coli ribosomal RNA operons in efficient 23 S rRNA synthesis in vivo.

A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16 S and 23 S genes of Escherichia coli ribosomal RNA operons. We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription. In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E. coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo. The rrnB mutant constructs contained an additional point mutation in each of the 16 S and 23 S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis. Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis. Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis. In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium. This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA. We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA.

Escherichia coli↗
PubMed2001

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable.

U2449 is one of many invariant residues in the central loop of domain V of 23S rRNA, a region that constitutes part of the peptidyltransferase center of the ribosome. In Escherichia coli, this U is post-transcriptionally modified to dihydrouridine (D) and is the only D modification found in E.coli rRNAs. To analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnB operon and assayed for their ability to support cell growth in a strain of E.coli lacking chromosomal rrn operons. Both purine substitution mutations were not viable. However, growth and antibiotic sensitivity of cells expressing only the mutant D2449C rRNA was indistinguishable from wild type. We conclude that while a pyrimidine is required at position 2449 for proper ribosomal function, the D modification is dispensable.

Base Sequence↗
PubMed1997

Evidence for transcriptional regulation of plastid photosynthesis genes in Arabidopsis thaliana roots.

Mechanisms underlying suppressed levels of transcripts for plastid photosynthesis genes in nongreen tissues such as roots and calli were analyzed in Arabidopsis thaliana, a plant suitable for further genetic dissection. A region encoding promoters of rbcL, the gene encoding the large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase, and the atpB/E operon for the beta and epsilon subunits of coupling factor one were cloned and sequenced. Transcripts for rbcL, atpB/E, and psbA, the gene for the D1 protein in the photosystem II reaction center, were barely detectable in roots of A. thaliana, whereas 16S rRNA was detected at a low level. The run-on transcription experiment revealed that expression of rbcL, atpB/E, and psbA was regulated at transcription. The copy number of plastid DNA in roots was one-fifth that in green leaves on the basis of total cellular DNA, suggesting that in the latter the DNA copy-number regulation also exists in plastid gene expression. Digestion of DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and subsequent polymerase chain reaction, as well as in vitro transcription of plastid DNAs with Escherichia coli RNA polymerase, resulted in no evidence of regulation by DNA modification. In spite of predominant suppression of expression of rbcL, atpB/E, and psbA at transcription in roots and calli, 16S rRNA levels were decreased because of low RNA stability.

Arabidopsis↗
PubMed1995

Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site.

Previous studies suggest that the mechanism of action of the ribosome in translation involves crucial transfer RNA (tRNA)-ribosomal RNA (rRNA) interactions. Here, a selection scheme was developed to identify bases in 16S rRNA that are essential for tRNA binding to the P site of the small (30S) ribosomal subunit. Modification of the N-1 and N-2 positions of 2-methylguanine 966 and of the N-7 position of guanine 1401 interfered with messenger RNA (mRNA)-dependent binding of tRNA to the P site. Modification of the same positions as well as of the N-1 and N-2 positions of guanine 926 interfered with mRNA-independent binding of tRNA at high magnesium ion concentration. These results suggest that these three bases are involved in intermolecular contacts between ribosomes and tRNA.

Aldehydes↗
PubMed2003

rRNA antitermination functions with heat shock promoters.

Transcription antitermination in the rRNA operons of Escherichia coli requires a unique nucleic acid sequence that serves as a signal for modification of the elongating RNA polymerase, making it resistant to Rho-dependent termination. We examined the antitermination ability of RNA polymerase elongation complexes that had initiated at three different heat shock promoters, dnaK, groE, and clpB, and then transcribed the antitermination sequence to read through a Rho-dependent terminator. Terminator bypass comparable to that seen with sigma(70) promoters was obtained. Lack of or inversion of the sequence abolished terminator readthrough. We conclude that RNA polymerase that uses sigma(32) to initiate transcription can adopt a conformation similar to that of sigma(70)-containing RNA polymerase, enabling it to interact with auxiliary modifying proteins and bypass Rho-dependent terminators.

Bacterial Proteins↗