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PubMed1994

New 18S.26S ribosomal RNA gene loci: chromosomal landmarks for the evolution of polyploid wheats.

Three new 18S.26S rRNA gene loci were identified in common wheat by sequential N-banding and in situ hybridization (ISH) analysis. Locus Nor-A7 is located at the terminal area of the long arm of 5A in both diploid and polyploid wheats. Locus Nor-B6 is located in N-band 1BL2.5 of the long arm of chromosome 1B in Triticum turgidum and Triticum aestivum. ISH sites, similar to Nor-B6, were also detected on the long arms of chromosomes 1G in Triticum timopheevii and 1S in Aegilops speltoides, but their locations on the chromosomes were different from that of Nor-B6, indicating possible chromosome rearrangements in 1GL and 1BL during evolution. The third new locus, Nor-D8, was only found on the short arm of chromosome 3D in the common wheat Wichita. The loss of rRNA gene locus Nor-A3 and gain of repetitive DNA sequence pSc119 on the terminal part of 5AS suggest a structural modification of 5AS. Comparative studies of the location of the 18S.26S rRNA gene loci in polyploid wheats and putative A and B (G) genome progenitor species support the idea that: (1) Triticum monococcum subsp. urartu is the donor of both the A and A(t) genome of polyploid wheats. (2) Ae. speltoides is closer to the B and G genome of polyploid wheats than Aegilops longissima and is the most probable progenitor of these two genomes.

Biological Evolution↗
PubMed2003

Identification of 13 novel human modification guide RNAs.

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.

Base Sequence↗
PubMed2006

Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution.

Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution and therefore were present in the last common ancestor. In this paper we report that KsgA orthologs from archaeabacteria and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life.

Amino Acid Motifs↗
PubMed1999

Novel predicted RNA-binding domains associated with the translation machinery.

Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.

Amino Acid Sequence↗
PubMed1988

The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein.

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.

Base Sequence↗
PubMed2003

Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center.

One of the oldest questions in RNA science is the role of nucleotide modification. Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications. Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding. Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations. Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others. The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed.

Base Sequence↗
PubMedDate not supplied

Antisignature oligonucleotides and their analogs as inhibitors of mollicutes-cofactors of HIV.

Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.

Acholeplasma laidlawii↗
PubMed2003

Ribosomal RNA processing and the role of SmMAK16 in ribosome biogenesis in Schistosoma mansoni.

Ribosome biogenesis is an essential and complex biological process with links to cell cycle control, DNA replication and aberrant cell growth. As the process becomes better understood at a mechanistic level, new opportunities arise to exploit its effects on maturation and fertility, two important targets in parasite therapeutics. While the physical structure and sequence of ribosomal RNA (rRNA) in the trematode Schistosoma mansoni have been described, the process of cleavage, modification and assembly of the mature ribonucleoprotein particles is not well characterized. We have investigated the cleavage pathway of rRNA in different life cycle stages of the parasite and present evidence supporting a proposed model. In addition, we have investigated the role of the S. mansoni nucleolar protein SmMAK16 in ribosome biogenesis. Co-immunoprecipitation data and polysome analysis support the hypothesis that SmMAK16 associates with pre-ribosomal precursor complexes. This work represents the first analysis of this important and complex process in schistosomes.

Animals↗
PubMed1998

rRNA chemical groups required for aminoglycoside binding.

Through an affinity chromatography based modification-interference assay, we have identified chemical groups within Escherichia coli 16S ribosomal RNA sequence that are required for binding the aminoglycoside antibiotic paromomycin. Paromomycin was covalently linked to solid support via a nine atom spacer from the 6"'-amine of ring IV, and chemical modifications to an A-site oligonucleotide that disrupted binding were identified. Positions in the RNA oligonucleotide that correspond to G1405(N7), G1491(N7), G1494(N7), A1408(N7), A1493(N7), A1408(N1), A1492(N1), and A1493(N1), as well as the pro-R phosphate oxygens of A1492 and A1493 in 16S rRNA are chemical groups that are essential for a high-affinity RNA-paromomycin interaction. These data are consistent with genetic, biochemical, and structural studies related to neomycin-class antibiotics and provide additional information for establishing an exact model for their interaction with the ribosome.

Adenosine↗
PubMed1996

RRP5 is required for formation of both 18S and 5.8S rRNA in yeast.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S) are synthesized as a single precursor which is subsequently processed into the mature rRNAs by a complex series of cleavage and modification reactions. In the yeast Saccharomyces cerevisiae, the early pre-rRNA cleavages at sites A0, A1 and A2, required for the synthesis of 18S rRNA, are inhibited in strains lacking RNA or protein components of the U3, U14, snR10 and snR30 small nucleolar ribonucleoproteins (snoRNPs). The subsequent cleavage at site A3, required for formation of the major, short form of 5.8S rRNA, is carried out by another ribonucleoprotein, RNase MRP. A screen for mutations showing synthetic lethality with deletion of the non-essential snoRNA, snR10, identified a novel gene, RRP5, which is essential for viability and encodes a 193 kDa nucleolar protein. Genetic depletion of Rrp5p inhibits the synthesis of 18S rRNA and, unexpectedly, also of the major short form of 5.8S rRNA. Pre-rRNA processing is concomitantly impaired at sites A0, A1, A2 and A3. This distinctive phenotype makes Rrp5p the first cellular component simultaneously required for the snoRNP-dependent cleavage at sites A0, A1 and A2 and the RNase MRP-dependent cleavage at A3 and provides evidence for a close interconnection between these processing events. Putative RRP5 homologues from Caenorhabditis elegans and humans were also identified, suggesting that the critical function of Rrp5p is evolutionarily conserved.

Alleles↗
PubMed1990

[Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)aminobenzylidene]m derivatives of oligodeoxyribonucleotides. IV. Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA].

By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV] followed by the RNase H treatment a number of 16S rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.

Alkylation↗
PubMed1997

The ribosomal 5.8S RNA as a target site for p53 protein in cell differentiation and oncogenesis.

Previous studies have shown that the ribosomal 5.8S rRNA of human cells is partially methylated in a tissue-specific fashion, a modification which occurs largely or entirely in the cytoplasm. More recent studies have shown that the 5.8S rRNA forms a covalent linkage with tumor suppressor p53 protein and have suggested that this RNA plays a functional role in protein elongation. We now show that the expression of p53 protein in Schizosaccharomyces pombe results in cells which: morphologically resemble transformed cells expressing mutant 5.8S rRNA; are equally compromised in their ability to sustain protein synthesis, in vitro; and contain polyribosome profiles which strongly resemble the elevated profiles which also are observed with mutated 5.8S rRNA. Taken together, these results provide new physiological evidence of the possibility that the 5.8S rRNA is an important target in the control of ribosome function during cell differentiation and oncogenesis.

Cell Differentiation↗
PubMed1975

Photoinduced convalent crosslinkage, in situ, of Escherichia coli 50 S ribosomal proteins to rRNA.

Irradiation of aqueous solutions of Escherichia coli 50 S ribosomal subunits with 253.7 nm light results in the covalent crosslinkage of the rRNA and protein components. Neither peptide bond cleavage nor protein-protein crosslinkage accompanies the crosslinkage reactionmin addition, substantial photoinduced modifications in the primary structure of the ribosomal proteins, other than crosslinkage to rRNA, are not detected. The crosslinkage of the ribosomal proteins to the rRNA proroceeds in two discrete, dose-dependent steps. The first step, requires an input of up to 3 with 10-20 Quanta of 253.7nm radiation, and results in the crosslinkage of less than half of the ribosomal proteins to the rRNA. The second step requires an input of greater than 3 with 10-20 Quanta of 253.7 nm radiation, and results in the crosslinkage of the remaining ribosomal proteins to the rRNA. The possible relationship of the nature of the corsslinkage reaction to the spatial orientations of the rRNA and protein molecules in the intact 50 S ribosomal subunits is discussed.

Bacterial Proteins↗
PubMed2001

Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA.

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.

Endoribonucleases↗
PubMed1977

Change of protein reactivity in mammalian ribosomal subunits as a function of temperature.

Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40 degrees C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67% of the lysine residues were methylated. Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37 degrees C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyl tRNA.

Animals↗
PubMed1982

Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3' end of tRNA.

A derivative of tRNAPhe carrying at its 3' end a photolyzable group was chemically synthesized by coupling p-azidobenzoylglycylhydrazide to periodate-oxidized tRNA. The reaction converts the 3'-terminal ribofuranoside residue into a six-membered ring. The binding of p-azidobenzoylglycylhydrazide-tRNA to 70-S ribosomes resembles the binding of Phe-tRNA in its requirement for poly(U) and Mg2+ dependence. Irradiation at wavelengths greater than 300 nm of complexes of p-azidobenzoylglycylhydrazide-tRNA and 70-S ribosomes results in the covalent binding of approximately 20% of the reversibly bound tRNA to the ribosomes. The labeling occurs predominantly at a single site in 23-S rRNA within the sequence included in the 18-S fragment. To identify the modified sequence, an RNase A digest of 23-S rRNA was fractionated on a hydrophobic matrix (Porapak Q) and adsorbed and non-adsorbed fractions were compared by fingerprint analysis. A relatively intense spot was observed in the expected region in the fingerprint of the adsorbed fraction. The presence of an unusual nucleotide in the sequence of this oligonucleotide was demonstrated by a mobility-shift analysis of a partial nuclease P1 digest. The sequence derived is G*-A-A-G-C (the asterisk denoting the site of modification). Sequences at six positions in the 23-S rRNA are compatible with the sequence of the oligonucleotide determined. Of the six positions five are located within nucleotides 1467-1887. This region of approximately 400 nucleotides is likely to include the sequence interacting with the 3' end of tRNA and possibly forming part of the peptidyl-transferase center.

Affinity Labels↗
PubMed2004

Macrolide resistance based on the Erm-mediated rRNA methylation.

Macrolide, lincosamide and streptogramin B (MLSB) antibiotics are extensively used for the treatment of wide variety of clinically important Gram-positive bacteria. MLSB antibiotics inhibit protein biosynthesis by targeting the peptidyl transferase centre within the 50S ribosomal subunit. The most widespread mechanism of bacterial resistance to MLSB antibiotics, reported early after their introduction into clinical practice is the modification of the target site exhibited by a family of rRNA methyltransferases designated Erm. Using S-adenosyl-L-methionine, Erm enzymes catalyze mono- or dimethylation of a specific adenine residue in the 23S rRNA. The methyl group sterically hinders the MLSB binding site and disrupts the hydrogen bonding between the macrolides and the rRNA, thus rendering bacteria resistant. This review summarizes the current understanding of Erm-mediated resistance, in light of high-resolution structural data of bacterial ribosome and with specific focus on the results of recent genetic, biochemical and structural studies of Erm methyltransferases and their cognate rRNA substrate. Although many features of MLSB resistance remain indistinct, the present knowledge can now serve as the guidance for development of both new antimicrobial drugs and potential inhibitors of Erm enzymes, hence providing a new lead to solve the urgent problem of the macrolide resistance based on the ribosome methylation.

Anti-Bacterial Agents↗
PubMed1999

ermSF, a ribosomal RNA adenine N6-methyltransferase gene from Streptomyces fradiae, confers MLS (macrolide-lincosamide-streptogramin B) resistance to E. coli when it is expressed.

The Erm family of methyltransferases confers the MLS antibiotic resistance to pathogenic microorganism through the mono- or dimethylation of a single adenine residue in 23S rRNA, which is known as the target site for modification. One of the erm genes, ermSF was cloned from Streptomyces fradiae NRRL 2702 by PCR and overexpressed in E. coli BL21(DE3) as both a soluble protein and insoluble aggregate (inclusion body) using the T7 promoter driven expression vector, pET23b. Even though most of the overexpressed protein existed as an inclusion body, E. coli cells showed resistance to erythromycin. The lowering of incubation temperature from 37 degrees C to 22 degrees C facilitated the purification of the protein by increasing the fraction of soluble protein. The soluble protein was purified using immobilized metal ion (Ni2+) affinity chromatography in a one-step manner to the apparent homogeneity. The 23S rRNA of E. coli was found to be a good substrate for the purified ErmSF.

Anti-Bacterial Agents↗