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PubMed2000

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides.

Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function. The goal of this study was to determine the functional significance of pairing interactions in the 690 region. Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies. To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected. A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced. Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides. Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function. We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699. Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions. The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch. Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure. NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair. Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation.

Base Pair Mismatch↗

PubMed1976

Photoinduced cross-linkage, in situ, of Escherichia coli 30S ribosomal proteins to 16S rRNA: identification of cross-linked proteins and relationships between reactivity and ribosome structure.

The kinetics of photoinduced cross-linkage of Escherichia coli 30S ribosomal proteins to the 16S-rRNA molecule in the intact Escherichia coli 30S ribosomal subunit was studied in this report. All of the 30S ribosomal proteins become cross-linked to the 16S rRNA before changes in the sedimentation characteristics of the 30S ribosomal subunit can be detected. The proteins exhibit different reactivities in the cross-linkage reaction. One group of proteins-S3, S7-S9, S11, S12, and S15-S19-is cross-linked to the 16S rRNA by single-hit kinetics, or by photoprocesses of nonunity but low multiplicities. A second group of proteins--S1, S2, S4-S6, S10, S13, S14, and S21--is cross-linked to the 16S rRNA by photoprocesses of a complex nature. A comparison of these data with other properties of the individual 30S ribosomal proteins related to ribosome structure indicated that most of the 30S ribosomal proteins cross-linked to the 16S rRNA by photoprocesses of low multiplicities had been classified rRNA-binding proteins by nonphotochemical methods, and most of the proteins cross-linked to the 16S rRNA by photoprocesses of large multiplicities had been classified as nonbinding proteins. There were certain exceptions to these correlations. Proteins S4 and S20, both RNA-binding proteins, become cross-linked to the 16S rRNA by photoprocessses of large multiplicities, and proteins S3, S11, S12, and S18, none of which have been classified RNA-binding proteins, exhibited low multiplicities in the cross-linkage reaction. All of these exceptions could be explained in terms of limitations inherent in the photochemical methods used in this study and in other types of methods that have been used to study RNA-protein interactions in the 30S ribosomal subunit. The data presented here also suggest that labile RNA-protein cross-links are present in the uv-irradiated 30S ribosomal subunits, and that neither peptide-bond cleavage nor photoinduced modification of the charged side-chain groups in the ribosomal proteins accompanied the cross-linkage reaction. However, some photoinduced RNA-chain breakage might have occurred.

Binding Sites↗

PubMed2005

Bacterial community structure in kimchi, a Korean fermented vegetable food, as revealed by 16S rRNA gene analysis.

Kimchi is a traditional Korean food fermented from a variety of vegetables. We elucidated the microbial community structure of five commercially produced kimchis made from Chinese cabbage by examining culture-independent 16S rRNA gene clone libraries. Most of the clones (347 out of 348) belonged to lactic acid bacteria and included several species of the genera Lactobacillus, Leuconostoc and Weissella. Weissella koreensis was found in all the samples and predominated in three of them (42.6-82%). Leuconostoc gelidum, Leuconostoc gasicomitatum and Lactobacillus sakei were common in the remaining kimchi clone libraries (>34%). The composition of bacterial phylotypes in kimchi varied between samples. Our approach revealed different community structures from those reported in previous culture-dependent studies based on phenotypic identification methods. The culture-independent method used here proved to be efficient and accurate and showed that the bacterial communities in kimchi differ from those in other fermented vegetable foods.

Base Sequence↗

PubMed1992

The structure of ribosomal protein S5 reveals sites of interaction with 16S rRNA.

Understanding the process whereby the ribosome translates the genetic code into protein molecules will ultimately require high-resolution structural information, and we report here the first crystal structure of a protein from the small ribosomal subunit. This protein, S5, has a molecular mass of 17,500 and is highly conserved in all lifeforms. The molecule contains two distinct alpha/beta domains that have structural similarities to several other proteins that are components of ribonucleoprotein complexes. Mutations in S5 result in several phenotypes which suggest that S5 may have a role in translational fidelity and translocation. These include ribosome ambiguity or ram, reversion from streptomycin dependence and resistance to spectinomycin. Also, a cold-sensitive, spectinomycin-resistant mutant of S5 has been identified which is defective in initiation. Here we show that these mutations map to two distinct regions of the molecule which seem to be sites of interaction with ribosomal RNA. A structure/function analysis of the molecule reveals discrepancies with current models of the 30S subunit.

Amino Acid Sequence↗

PubMed1994

Characterization of a nucleolar 2'-O-methyltransferase and its involvement in the methylation of mouse precursor ribosomal RNA.

A nucleolar 2'-O-methyltransferase, partially purified from isolated mouse nucleoli, catalyzes the methylation of each of the four nucleosides, although to different levels depending on the RNA substrate. Similar to most methyltransferases which use S-adenosyl-L-methionine (SAM) as the methyl donor, the nucleolar 2'-O-methyltransferase was shown to bind S-adenosyl-L-homocysteine (SAH) (Kd = 0.17 microM), a product of the transfer reaction, as tightly as SAM (Kd = 0.24 microM). Binding assays also demonstrated stereospecificity about the sulfonium center of SAM. The naturally occurring S-chiral form of SAM had a 10-fold higher binding affinity than the R-chiral form. In addition, the alpha-amino group of the methionine moiety and the 6-amino group of the adenine moiety were shown to be required for maximal binding. The relative high affinity for both SAM and SAH may reflect a mechanism by which ribosome biogenesis is, in part, coordinated with cell growth, since a decrease in SAM:SAH ratio correlates with decreasing levels of 2'-O-methylation. The availability of unmethylated, in vitro-derived rRNA transcripts has made it possible to explore questions relating to the specificity for the RNA substrate. Using an in vitro-derived 28S rRNA transcript, the enzyme selectively methylated the sequence AmGmCm that occurs in a single-stranded bridge spanning two highly conserved structural domains of 28S rRNA. These results demonstrated that the purified nucleolar 2'-O-methyltransferase was sufficient to accurately methylate this region of 28S rRNA, and were taken to support the involvement of this nucleolar enzyme in the posttranscriptional methylation of the 47S precursor ribosomal RNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1995

Mycoplasma gallisepticum 16S rRNA genes.

The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not possess the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA.

Base Sequence↗

PubMed2005

Predicting rRNA-, RNA-, and DNA-binding proteins from primary structure with support vector machines.

In the post-genome era, the prediction of protein function is one of the most demanding tasks in the study of bioinformatics. Machine learning methods, such as the support vector machines (SVMs), greatly help to improve the classification of protein function. In this work, we integrated SVMs, protein sequence amino acid composition, and associated physicochemical properties into the study of nucleic-acid-binding proteins prediction. We developed the binary classifications for rRNA-, RNA-, DNA-binding proteins that play an important role in the control of many cell processes. Each SVM predicts whether a protein belongs to rRNA-, RNA-, or DNA-binding protein class. Self-consistency and jackknife tests were performed on the protein data sets in which the sequences identity was < 25%. Test results show that the accuracies of rRNA-, RNA-, DNA-binding SVMs predictions are approximately 84%, approximately 78%, approximately 72%, respectively. The predictions were also performed on the ambiguous and negative data set. The results demonstrate that the predicted scores of proteins in the ambiguous data set by RNA- and DNA-binding SVM models were distributed around zero, while most proteins in the negative data set were predicted as negative scores by all three SVMs. The score distributions agree well with the prior knowledge of those proteins and show the effectiveness of sequence associated physicochemical properties in the protein function prediction. The software is available from the author upon request.

Amino Acid Sequence↗

PubMed2004

The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059.

Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.

Anti-Bacterial Agents↗

PubMed2001

Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene.

The microbial community structure of granular sludge from an upflow anaerobic sludge blanket (UASB) reactor treating brewery effluent was studied by denaturing gradient gel electrophoresis (DGGE). Twelve major bands were observed in the DGGE fingerprint for the Bacteria domain and four bands for the Archaea domain. Of the bacterial bands observed, six were successfully purified and sequenced. Among them, three were related to the gram-positive low G + C group, one to the Delta subclass of the Proteobacteria, one to the Gamma subclass, and one to the Cytophaga group with no close related sequence. The 16S rRNA sequences of the four archaeal bands were closely associated with Methanosaeta concilii and Methanobacterium formicum.

Bacteria, Anaerobic↗

PubMed1996

Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA.

Using 5' end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro replication products indicated that templates from either of the origin regions supported replication, while the vector alone or ctDNA clones from other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer extension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5' end of the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were insensitive to both alkali and RNase treatment, suggesting that RNA primers had already been removed from the 5' end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.

Base Composition↗

PubMed2002

Protein synthetic machinery in the dendrites of the magnocellular neurosecretory neurons of wild-type Long-Evans and homozygous Brattleboro rats.

There is growing evidence of local protein synthesis in neuronal dendrites, especially in relation to synaptic activity. The hypothalamic magnocellular system is a robust model for peptidergic neurons, especially for the study of dendrites. Quantitative electron microscopy, immunocytochemistry and non-radioactive in situ hybridization (with tyramide signal amplification) were used to compare dendrites of magnocellular neurons in the supraoptic nucleus of wild-type rats and of homozygous Brattleboro (BB) rats which are subject to long-term hyper-osmotic stimulation because they cannot secrete vasopressin. The dendrites contained free polyribosomes, cisterns of rough endoplasmic reticulum (ER) and small Golgi-like elements. These were clustered in the dendrites, mostly near the plasma membrane. All were increased in amount in the enlarged dendrites of the BB rats. The presence of polyribosomes and cisterns of rER implies that both cytosolic and membrane-inserting proteins are synthesized in the dendrites. The ER marker protein disulfide isomerase extended far into dendrites, but Golgi element markers (mid-Golgi and trans-Golgi network) were distributed mainly in their proximal parts. In BB rats, all the labeling was stronger. 28S rRNA, initiator tRNA(Met), and poly(A) mRNA were revealed extending into proximal and middle parts of dendrites where intensely reactive punctate structures were common. 28S rRNA could be detected in the distal parts of the dendrites. The length of positively stained dendrites was increased significantly for all these RNAs in BB rats. The results provide morphological evidence that magnocellular dendrites have the capacity for local protein syntheses and that this is increased in chronic hyperosmotic stress.

Animals↗

PubMed1997

A region containing repeated elements is associated with transcriptional termination of Leishmania infantum ribosomal RNA genes.

A novel repetitive DNA element has been isolated from the Leishmania infantum genome. The 348 bp long element, designated LiR3, was found to be located downstream from the 3'-end of the ribosomal RNA (rRNA) genes. This LiR3 element has short sequences with potential to form stem-loop structures similar to those of the bacterial rho-independent transcriptional terminators. Given both the structural features and the genomic location of this element we searched for a possible functional implication of these structures in the termination of rRNA transcription. Nuclear run-on assays indicated that indeed there is a transcriptional blockage associated with the LiR3 element. Several chi-like elements, resembling the recombination-promoting sites of Escherichia coli, were identified within the sequences associated with the stem-loop structures. A possible implication of these chi-like elements in rRNA gene conversion events is discussed.

Animals↗

PubMed2002

Novel mitochondrial 16S rRNA mutation, 3200T-->C, associated with adult-onset type 2 diabetes.

OBJECTIVE: To investigate the role of a potential diabetes-related mitochondrial region, which includes two previously reported mutations, 3243A-->G and 3316G-->A, in Chinese patients with adult-onset type 2 diabetes. METHODS: A total of 277 patients and 241 normal subjects were recruited for the study. Mitochondrial nt 3116 - 3353, which spans the 16S rRNA, tRNA(leu(UUR)) and the NADH dehydrogenase 1 gene, were detected using polymerase chain reaction (PCR), direct DNA sequencing, PCR-restriction fragment length polymorphism and allele-specific PCR. Variants were analyzed by two-tailed Fisher exact test. The function of the variants in 16S rRNA were predicted for minimal free energy secondary structures by RNA folding software mfold version 3. RESULTS: Four homoplasmic nucleotide substitutions were observed, 3200T-->C, 3206C-->T, 3290T-->C and 3316G-->A. Only the 3200T-->C mutation is present in the diabetic population and absent in the control population. No statistically significant associations were found between the other three variants and type 2 diabetes. The 3200T-->C and 3206C-->T nucleotide substitutions located in 16S rRNA are novel variants. The 3200T-->C caused a great alteration in the minimal free energy secondary structure model while the 3206C-->T altered normal 16S rRNA structure little. CONCLUSIONS: The results suggest that the 3200T-->C mutation is linked to the development of type 2 diabetes, but that the other observed mutations are neutral. In contrast to the Japanese studies, the 3316G-->A does not appear to be related to type 2 diabetes.

Age of Onset↗

PubMed1993

Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation.

The postulated central pseudoknot formed by regions 9-13/21-25 and 17-19/916-918 of 16S rRNA of Escherichia coli is phylogenetically conserved in prokaryotic as well eukaryotic species. This pseudoknot is located at the center of the secondary structure of the 16S rRNA and connects the three major domains of this molecule. We have introduced mutations into this pseudoknot by changing the base-paired residues C18 and G917, and the effect of such mutations on the ribosomal activity was studied in vivo, using a 'specialized' ribosome system. As compared with ribosomes having the wild-type pseudoknot, the translational activity of ribosomes containing an A, G or U residue at position 18 was dramatically reduced, while the activity of mutant ribosomes having complementary bases at positions 18 and 917 was at the wild-type level. The reduced translational activity of those mutants that are incapable of forming a pseudoknot was caused by their inability to form 70S ribosomal complexes. These results demonstrate that the potential formation of a central pseudoknot in 16S rRNA with any base-paired residues at positions 18 and 917 is essential to complete the initiation process.

Base Composition↗

PubMed1981

Cloning and characterization of ribosomal RNA gene of Physarum polycephalum.

As a first step in studies on the structure and expression of rRNA gene of Physarum polycephalum, the cloning of the gene in Escherichia coli with plasmid vector DNAs was performed and 8 different kinds of clones containing 26S, 19S, and 5.85 rRNA genes were obtained. Using these cloned fragments, the location of these rRNA genes was determined by the Southern hybridization and S1 mapping method. Cloning of rDNA fragments made it possible to analyze the fine structure of the rDNA.

Cloning, Molecular↗

PubMed2004

The 18S rRNA from Odontophrynus americanus 2n and 4n (Amphibia, Anura) reveals unusual extra sequences in the variable region V2.

The nucleotide sequence of the rDNA 18S region isolated from diploid and tetraploid species of the amphibian Odontophrynus americanus was determined and used to predict the secondary structure of the corresponding 18S rRNA molecules. Comparison of the primary and secondary structures for the 2n and 4n species confirmed that these species are very closely related. Only three nucleotide substitutions were observed, accounting for 99% identity between the 18S sequences, whereas several changes were detected by comparison with the Xenopus laevis 18S sequence (96% identity). Most changes were located in highly variable regions of the molecule. A noticeable feature of the Odontophrynus 18S rRNA was the presence of unusual extra sequences in the V2 region, between helices 9 and 11. These extra sequences do not fit the model for secondary structure predicted for vertebrate 18S rRNA.

Animals↗

PubMed1997

Structural features of the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA defined using NMR spectroscopy.

Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597 x C643 and A596 x U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595 x (A596 x U644). The NMR data suggest that S8-RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598 x A640 base pair and appears to stabilize the G597 x C643 and A596 x U644 base pairs.

Amino Acid Sequence↗

PubMed1983

Effect of 2'-O-methylation on the structure of mammalian 5.8S rRNAs and the 5.8S-28S rRNA junction.

The mammalian 5.8S rRNA contains a partially 2'-O-methylated uridylic acid residue at position 14 which is largely or entirely methylated in the cytoplasm (Nazar, R.N., Sitz, T.O. and Sommers, K.D. (1980) J. Mol. Biol. 142, 117-121). The effect of this methylation on the 5.8S RNA structure and 5.8-28S rRNA junction was investigated using both chemical and physical approaches. Electrophoretic studies indicated that the free 5.8S rRNA can take on at least two different conformations and that the 2'-O-methylation at U14 restricts the molecule to the more hydrodynamically open form. Structural studies using limited pancreatic or T1 ribonuclease digestion indicated that the methylated conformation was more susceptible to digestion, consistent with a more open tertiary structure. Modification-exclusion studies indicated that the first 29 nucleotides at the 5' end and residues 140 through 158 at the 3' end affect the 5.8S-28S rRNA interaction, supporting previous suggestions that the 5.8S RNA interacts with its cognate high molecular weight component through its termini. These results also suggested that the 2'-O-methylated uridylic acid residue plays a role in the 5.8S-28S rRNA interaction and thermal denaturation studies confirmed this by showing that methylation destabilizes the 5.8S-28S rRNA junction. The 5.8-28S rRNA interaction appears to be more complex than previously believed.

Animals↗