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At least 397 records · Page 22Linked to original sources
PubMed1985

Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA.

The site of Escherichia coli 16S ribosomal RNA cross-linked to the 5'-anticodon base of A site bound E. coli valyl-tRNA was identified. Cross-linking was via the affinity probe 6-[(2-nitro-4-azidophenyl)amino]caproate (NAK) or 3-[[2-[(2-nitro-4-azidophenyl)amino]ethyl]dithio]propionate (SNAP) attached to the carboxyl group of the 5'-anticodon base 5-(carboxyethoxy)uridine via an ethylenediamine spacer [Gornicki, P., Ciesiolka, J., & Ofengand, J. (1985) Biochemistry (preceding paper in this issue)]. With both probes, RNase T1 digestion of the isolated 16S RNA-tRNA covalent complex, 5'-32P postlabeling, and gel electrophoresis yielded two oligonucleotides larger than any fragments from non-cross-linked tRNA or rRNA. Appearance of the oligomers was dependent on the presence of the probe on the tRNA. Unmodified tRNA in the A and/or P sites did not yield any product. The presence of elongation factor Tu in the incubation mixture was also required. Dithiothreitol (DDT) treatment of the SNAP-induced covalent complex prior to electrophoresis also abolished the oligomers. Only the larger of the two oligomers (present in a 3:1 ratio) was sequenced. The SNAP dimer was cleaved with DTT, and the rRNA and tRNA oligomers were separated and sequenced as monomers. The NAK dimer was sequenced without cleavage by taking advantage of the differences in electrophoretic mobility among sequence and/or composition isomers of the same length. In both cases, the rRNA oligomer was identified as UACACACCG1401, and the nucleotide cross-linked was shown to be the C1400 residue. The expected tRNA modification site was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed2001

Macrolide resistance mechanisms in Gram-positive cocci.

Two principal mechanisms of resistance to macrolides have been identified in Gram-positive bacteria. Erythromycin-resistant methylase is encoded by erm genes. Resultant structural changes to rRNA prevent macrolide binding and allow synthesis of bacterial proteins to continue. Presence of the erm gene results in high-level resistance. Modification of the mechanism whereby antibiotics are eliminated from the bacteria also brings about resistance. Bacteria carrying the gene encoding macrolide efflux (i.e. the mefE gene) display relatively low-level resistance. Azithromycin, because of its ability to achieve concentrations at sites of infections, is capable of eradicating mefE-carrying strains. Other resistance mechanisms, involving stimulation of enzymatic degradation, appear not to be clinically significant.

Anti-Bacterial Agents↗
PubMed1981

The proteins of the messenger RNA binding site of Escherichia coli ribosomes.

Oligo(U) derivatives with [14C]-4-(N-2-chloroethyl-N-methylamino)benzaldehyde attached to 3'-end cis-diol group via acetal bond, p(Up)n-1UCHRCl as well as with [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to 5'-phosphate via amide bond, ClRCH2NHpU(pU)6 were used to modify 70S E. coli ribosomes near mRNA binding centre. Within ternary complex with ribosome and tRNAPhe all reagents covalently bind to ribosome the extent of modification being 0.1-0.4 mole/mole 70S. p(Up)n-1UCHRCl alkylates either 30S (n=5,7) or both subunits (n=6,8). rRNA is preferentially modified within 30S subunit. ClRCH2NHpU(pU)6 alkylates both subunits the proteins being mainly modified. The distribution of the label among proteins differ for various reagents. S4, S5, S7, S9, S11, S13, S15, S18 and S21 are found to be alkylated within 30S subunit, proteins L1, L2, L6, L7/L12, L19, L31 and L32 are modified in the 50S subunit. Most proteins modified within 30S subunit are located at the "head" of this subunit and proteins modified within 50S subunit are located at the surface of the contact between this subunit and the "head" of 30S subunit at the model of Stoffler.

Benzaldehydes↗
PubMedDate not supplied

[Protein environment of the sense codon of the template in the A site of the human ribosome as inferred from crosslinking to oligoribonucleotide derivatives].

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA(Phe), which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA(Phe) and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic ribosomes.

Codon↗
PubMed1995

Changing nucleosome positions through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

Base Sequence↗
PubMed1994

Chemical secondary structure probing of two highly methylated regions in Xenopus laevis 28S ribosomal RNA.

The large ribosomal subunit (LSU) RNA or 28S rRNA of vertebrates is characterized by two highly conserved and methylated regions towards the 3' end of the molecule that extend from domains IV to V of the molecule. In this report we describe the probing of the secondary structure of these two highly methylated regions in Xenopus laevis LSU RNA by chemical modification using the single-strand nucleotide specific probes; dimethyl sulphate (DMS) and 1-cyclo-hexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulphonate (CMCT) followed by primer extension. The same regions in in vitro synthesized unmethylated X. laevis 28S rRNA were also probed for comparison. Our results in general tend to support the theoretically determined secondary structure model for the probed domains. From the results obtained, methylated cellular LSU RNA appears to be relatively more reactive than the in vitro transcript to the chemical probes. Accessibility to the probes was found to be similar at most sites for cellular and in vitro transcript LSU RNAs. This implies that structural destabilization due to 2'-O-methylations in cellular LSU RNA is not significant.

Animals↗
PubMed1991

Parallel origins of duplications and the formation of pseudogenes in mitochondrial DNA from parthenogenetic lizards (Heteronotia binoei; Gekkonidae).

Analysis of mitochondrial DNAs (mtDNAs) from parthenogenetic lizards of the Heteronotia binoei complex with restriction enzymes revealed an approximately 5-kb addition present in all 77 individuals. Cleavage site mapping suggested the presence of a direct tandem duplication spanning the 16S and 12S rRNA genes, the control region and most, if not all, of the gene for the subunit 1 of NADH dehydrogenase (ND1). The location of the duplication was confirmed by Southern hybridization. A restriction enzyme survey provided evidence for modifications to each copy of the duplicated sequence, including four large deletions. Each gene affected by a deletion was complemented by an intact version in the other copy of the sequence, although for one gene the functional copy was heteroplasmic for another deletion. Sequencing of a fragment from one copy of the duplication which encompassed the tRNA(leu)(UUR) and parts of the 16S rRNA and ND1 genes, revealed mutations expected to disrupt function. Thus, evolution subsequent to the duplication event has resulted in mitochondrial pseudogenes. The presence of duplications in all of these parthenogens, but not among representatives of their maternal sexual ancestors, suggests that the duplications arose in the parthenogenetic form. This provides the second instance in H. binoei of mtDNA duplication associated with the transition from sexual to parthenogenetic reproduction. The increased incidence of duplications in parthenogenetic lizards may be caused by errors in mtDNA replication due to either polyploidy or hybridity of their nuclear genomes.

Amino Acid Sequence↗
PubMedDate not supplied

[Affinity modification of Escherichia coli ribosomes by 4-[(N-2-chloroethyl)N-methylamino]benzyl-5'-phosphamide hexauridylate in a complex stabilized by codon-anticodon interactions on P and A sites].

Affinity labeling of E. coli ribosomes with 4-[(N-2-chloroethyl)-N-methylamino] benzyl-5'-phosphamide of hexauridylate was studied within the complex containing tRNAPhe at P site and Phe-tRNAPhe at A site directed by EF-Tu and GTP. Ribosomal proteins as well as rRNA both in 30S and 50S subunits were found to be labelled within the complex. Labeled proteins were identified as S3, S9 and L2. Selectivity of affinity labeling with mRNA analogs was shown to depend on the functional state of the ribosomes. Modification was more selective within the complex stabilized by codon-anticodon interaction both at A and P-sites than within the complex in which this interaction takes place preferentially at P site.

Anticodon↗
PubMed1995

Phylogeny of Eucalyptus and Angophora based on 5S rDNA spacer sequence data.

Sequences of the 5S rDNA repeat have been determined from four outgroup taxa (Arillastrum group), 2 Angophora species, and 19 Eucalyptus species (including representatives from all informal subgenera). Alignment shows that, as in other plants, the tandemly repeated 5S rRNA genes are highly conserved while the noncoding intergenic spacers are variable. A 50-base pair (bp) repeating element, which has undergone duplication and modification in certain taxa, is identifiable within the spacer and accounts for much of the variability. There were several other multiresidue deletion/insertion events in the spacers that define major clades. Based on the indel events and modifications of the 50-bp element it is apparent that the spacer from bloodwood (informal subgenera Blakella and Corymbia) species of Eucalyptus is more similar to that of Angophora than to non-bloodwood species of Eucalyptus. Parsimony analysis showed Eucalyptus to be paraphyletic, with the bloodwoods being more closely related to Angophora than to the rest of Eucalyptus. This finding supports taxonomic revision of Eucalyptus.

Australia↗
PubMed2005

The GTP binding protein Obg homolog ObgE is involved in ribosome maturation.

Obg proteins belong to a subfamily of GTP binding proteins, which are highly conserved from bacteria to human. Mutations of obgE genes cause pleiotropic defects in various species but the function remained unclear. Here we examine the function of ObgE, the Obg homolog in Escherichia coli. The growth rate correlates with the amount of ObgE in cells. Co-fractionation experiments further suggest that ObgE binds to 30S and 50S ribosomal subunits, but not to 70S ribosome. Pull-down assays suggest that ObgE associates with several specific ribosomal proteins of 30S and 50S subunits, as well as RNA helicase CsdA. Purified ObgE cosediments with 16S and 23S ribosomal RNAs in vitro in the presence of GTP. Finally, mutation of ObgE affects pre-16Sr-RNA processing, ribosomal protein levels, and ribosomal protein modification, thereby significantly reducing 70S ribosome levels. This evidence implicates that ObgE functions in ribosomal biogenesis, presumably through the binding to rRNAs and/or rRNA-ribosomal protein complexes, perhaps as an rRNA/ribosomal protein folding chaperone or scaffold protein.

Cytosol↗
PubMed2001

Review of macrolides and ketolides: focus on respiratory tract infections.

The first macrolide, erythromycin A, demonstrated broad-spectrum antimicrobial activity and was used primarily for respiratory and skin and soft tissue infections. Newer 14-, 15- and 16-membered ring macrolides such as clarithromycin and the azalide, azithromycin, have been developed to address the limitations of erythromycin. The main structural component of the macrolides is a large lactone ring that varies in size from 12 to 16 atoms. A new group of 14-membered macrolides known as the ketolides have recently been developed which have a 3-keto in place of the L-cladinose moiety. Macrolides reversibly bind to the 23S rRNA and thus, inhibit protein synthesis by blocking elongation. The ketolides have also been reported to bind to 23S rRNA and their mechanism of action is similar to that of macrolides. Macrolide resistance mechanisms include target site alteration, alteration in antibiotic transport and modification of the antibiotic. The macrolides and ketolides exhibit good activity against gram-positive aerobes and some gram-negative aerobes. Ketolides have excellent activity versus macrolide-resistant Streptococcus spp. Including mefA and ermB producing Streptococcus pneumoniae. The newer macrolides, such as azithromycin and clarithromycin, and the ketolides exhibit greater activity against Haemophilus influenzae than erythromycin. The bioavailability of macrolides ranges from 25 to 85%, with corresponding serum concentrations ranging from 0.4 to 12 mg/L and area under the concentration-time curves from 3 to 115 mg/L x h. Half-lives range from short for erythromycin to medium for clarithromycin, roxithromycin and ketolides, to very long for dirithromycin and azithromycin. All of these agents display large volumes of distribution with excellent uptake into respiratory tissues and fluids relative to serum. The majority of the agents are hepatically metabolised and excretion in the urine is limited, with the exception of clarithromycin. Clinical trials involving the macrolides are available for various respiratory infections. In general, macrolides are the preferred treatment for community-acquired pneumonia and alternative treatment for other respiratory infections. These agents are frequently used in patients with penicillin allergies. The macrolides are well-tolerated agents. Macrolides are divided into 3 groups for likely occurrence of drug-drug interactions: group 1 (e.g. erythromycin) are frequently involved, group 2 (e.g. clarithromycin, roxithromycin) are less commonly involved, whereas drug interactions have not been described for group 3 (e.g. azithromycin, dirithromycin). Few pharmacoeconomic studies involving macrolides are presently available. The ketolides are being developed in an attempt to address the increasingly prevalent problems of macrolide-resistant and multiresistant organisms.

Adolescent↗
PubMed2006

Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations.

Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

Base Sequence↗
PubMed1989

Glucocorticoid-induced stimulation of ribosomal gene transcription in rat hepatoma cells is mediated by modification of RNA polymerase I or an associated factor.

An in vitro transcription system was developed from H411EC3 (H4) hepatoma cells, which mimics the in vivo up-regulation by glucocorticoid hormones on ribosomal RNA (rRNA) synthesis. Ribosomal DNA (rDNA) transcription in extracts derived from H4 cells grown in the presence of 100 nM triamcinolone acetonide was 4- to 5-fold greater than that in extracts derived from cells grown in the absence of glucocorticoid. This effect was not a general stimulation by the steroid, as RNA polymerase II transcription of the metallothionein-1 gene which lacked a glucocorticoid responsive element was unaffected. The increased transcription in hormone-treated extracts was also independent of differential ribonuclease activities or inhibitors as ascertained by the inclusion of ribonuclease inhibitor and mixing experiments, respectively. Chromatography of H4 cell extracts on heparin-sepharose followed by transcription complementation analysis, showed that the hormone-induced stimulatory activity eluted with the fraction (TFIA) which contains RNA polymerase I (Pol I). Immunoblot analysis with specific anti-Pol I antibody showed similar subunit profiles in the absence and presence of the hormone. The presence of a Pol I enhancer element in addition to the rDNA promoter did not further modify the glucocorticoid-induced transcription. These results indicate that the glucocorticoid-mediated effects could be observed in cell extracts which accurately initiate transcription of cloned rat rDNA. Moreover, the alterations of rDNA transcription by the hormone is effected by a factor which elutes with fraction TFIA.

Animals↗
PubMed2007

Modifications of aminoglycoside antibiotics targeting RNA.

The increased awareness of the central role of RNA has led to realization that RNA, as structural and functional information accumulation, is also drug target to small molecular therapy. Aminoglycosides are a group of well-known antibiotics, which function through binding to specific sites in prokaryotic ribosomal RNA (rRNA) and affecting the fidelity of protein synthesis. Unfortunately, their clinical practice has been curtailed by toxicity and rapid increasing number of resistant strains. Therefore, it is highly desirable to design new modified aminoglycosides that will overcome the undesirable properties of natural occurring aminoglycosides. On the other hand, aminoglycosides as potential antiviral (HIV) agents were also reported. Herein, we survey the current efforts to develop new aminoglycoside derivatives with modification and reconstruction on each sugar ring and review the latest advances in structure-activity relationships (SAR).

Aminoglycosides↗
PubMed1989

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.

Base Sequence↗
PubMed1971

Effect of a modified separation procedure on the size and protein-synthetic activity of membrane-bound liver polyribosomes.

1. Various subcellular fractions containing ribosomes were isolated from rat liver. 2. In the presence of [(14)C]leucine and Sephadex-treated cell sap the radioactivity incorporated into the synthesized protein resulting from the incubation of microsomal preparations or deoxycholate-treated polyribosomes was dependent on the amount of rRNA incubated. In contrast, when Sephadex-treated post-mitochondrial supernatant was incubated, the radioactivity incorporated into the synthesized protein was independent of the amount of rRNA incubated. 3. Microsomal preparations and membrane-bound ribosomes, prepared by the standard procedure, incorporated less [(14)C]leucine into protein, per mg of rRNA incubated, than free or deoxycholate-treated polyribosomes; accordingly, polyribosomes associated with the former fractions were found mainly as monomers. 4. If microsomal fractions or membrane-bound ribosomes were prepared by a simple modification of the standard procedure, i.e. by centrifugation on to a ;cushion' of 2m-sucrose, their protein-synthesizing activity was of the same order as that of the original post-mitochondrial supernatant, and membrane-free and deoxycholate-treated polyribosomes; in this case polyribosome profiles showed that very little degradation had occurred and compared well with those obtained for post-mitochondrial supernatant and isolated polyribosomes. 5. A method is described (Appendix) that provides a rapid and reliable assessment of the concentration of rRNA in subcellular fractions.

Animals↗
PubMed1999

Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli.

Three ribosomal RNA mutations conferring resistance to the antibiotic kasugamycin were isolated using a strain of Escherichia coli in which all of the rRNA is transcribed from a plasmid-encoded rrn operon. The mutations, A794G, G926A, and A1519C, mapped to universally conserved sites in the 16 S RNA gene. Site-directed mutagenesis studies showed that virtually all mutations at these three sites conferred kasugamycin resistance and had very slight effects on cell growth. It has been known for many years that the absence of post-transcriptional modification at A1519 and the adjacent A1518 in strains lacking a functional KsgA methylase produces a kasugamycin resistance phenotype. Mutations at A1519 conferred kasugamycin resistance and had minor effects on cell growth, whereas mutations at 1518 did not confer resistance and increased the doubling time of the cells dramatically. Expression of mutations at A1518/A1519 in a methylase deficient ksgA(-)strain had divergent effects on the phenotype of the rRNA mutants, suggesting that the base identity at either position does not affect methylation at the adjacent site. Residues A794 and G926 are protected from chemical modification by kasugamycin and tRNA, and have been implicated in the initiation of protein synthesis. Despite the universal conservation and functional importance of these residues, the results presented here show that the identity of the bases is not critical for ribosomal function.

Aminoglycosides↗
PubMed1997

Structural modifications of RNA influence the 5' exoribonucleolytic hydrolysis by XRN1 and HKE1 of Saccharomyces cerevisiae.

Two 5' exoribonucleases, XRN1 and HKE1, of Saccharomyces cerevisiae have been found to have very important cellular roles, XRN1 playing a key role in mRNA turnover and HKE1 in pre-rRNA processing. Here, an analysis of strong secondary structures in RNA that cause blocks or stalls (accumulation of RNA fragments that are shortened from the 5' end to the site of the secondary structure insertion) in the processive exoribonucleolytic hydrolysis reactions is reported. With both enzymes, oligo(G) tracts of lengths 18, 16, and 9 stall quite effectively, and the stalls are close to the start of the oligo(G) stretch. Two strong stem-loop structures cause measurable but low-level stalls with both enzymes. If the stem-loop structure is placed close to the 5' end of the RNA, substantial inhibition of overall RNA hydrolysis occurs with HKE1 and less, but measurable, inhibition with XRN1. RNA structural modification caused by protein complexing has been investigated by using poly(A) binding protein. The hydrolysis of poly(A) by XRN1 is inhibited by poly(A) binding protein, while HKE1 activity is not affected.

Base Sequence↗