Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “rRNA Operon”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources
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↗
PubMed2000

Crystal structure of ribosomal protein L4 shows RNA-binding sites for ribosome incorporation and feedback control of the S10 operon.

Ribosomal protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and proteins L2 and L3, actively participate in the catalysis of peptide bond formation. Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon. The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the protein with an alternating alpha/beta fold and a large disordered loop region. Two separate binding sites for RNA are discernible. The N-terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking alpha-helices. The C-terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non-consecutive alpha-helices. The structure also suggests a C-terminal protein-binding interface, through which L4 could be interacting with protein components of the transcriptional and/or translational machineries.

Amino Acid Sequence↗
PubMed1991

Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207.

Mutations were constructed at three sites in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680. The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation. We constructed a deletion mutation at C967 (delta 967) and three substitution mutations at each of the following sites: G966, C967, and G1207. By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes. We found that delta 967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate. The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate. These results help to establish the importance of methyl-modified regions to ribosome function.

Base Sequence↗
PubMed2005

Ecotypes of planktonic actinobacteria with identical 16S rRNA genes adapted to thermal niches in temperate, subtropical, and tropical freshwater habitats.

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6 degrees C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.

Actinobacteria↗
PubMed2000

Translational defects of Escherichia coli mutants deficient in the Um(2552) 23S ribosomal RNA methyltransferase RrmJ/FTSJ.

We recently identified RrmJ (alias FtsJ), the first encoded protein of the rrmJ-hflB heat shock operon, as an Um(2552) methyltransferase of the 23S rRNA. We now report that the rrmJ-deficient strain exhibits growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S(30) rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity. Polysome profile analysis of the rrmJ strain reveals an increase in the proportion of free 30S and 50S subunits at both 30 and 42 degrees C. These results suggest that the RrmJ-catalyzed methylation of Um(2552) in 23S RNA strengthens ribosomal subunit interactions, increases protein synthesis activity, and improves cell growth rates even at non-heat shock temperatures.

Anti-Bacterial Agents↗
PubMed1997

Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms.

Base Sequence↗
PubMed2000

PCR amplification and phylogenetic analysis of groESL operon sequences from Ehrlichia ewingii and Ehrlichia muris.

Broad-range PCR primers were used to amplify part of the groESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.

Animals↗
PubMed1990

Effects of mutagenesis of C912 in the streptomycin binding region of Escherichia coli 16S ribosomal RNA.

Four different mutations were produced at position 912 of Escherichia coli 16S rRNA in the multicopy plasmid pKK3535. Cells transformed with the mutant plasmids were assayed for growth in steptomycin. The U912 mutant conferred low level streptomycin resistance as reported originally by Montandon and co-workers (EMBO J 1986; 5:3705-3708). The G912 mutant also gave low level resistance but, unlike U912, caused significant retardation in growth rate and tended to select for fast-growing revertants. The A912 mutant was without effect on growth rate or streptomycin sensitivity, while deletion of C912 was lethal. Cells with U912 were selected for increased streptomycin resistance (MIC up to 160 micrograms/ml) and then cured of the plasmid. The cured cells retained a higher level of streptomycin resistance (MIC: 80 micrograms/ml) than the original wild type strain (MIC: 10 micrograms/ml), but sequencing by reverse transcriptase showed no evidence of U912 in the cellular 16S rRNA. Thus, recombination of the plasmid-coded U912 mutation into host rrn operons was not the mechanism by which increased streptomycin resistance occurred. The plasmid with U912 was transformed into three different streptomycin-dependent strains to determine whether the rRNA mutation, which presumably alters streptomycin binding, was compatible with S12 mutations which require bound streptomycin in order to function properly. In one strain, no transformants could be isolated, indicating that the plasmid was lethal. The two other streptomycin-dependent strains were transformed, but ribosomes containing the mutant rRNA were non-functional.

Base Sequence↗
PubMedDate not supplied

Genetic probes of ribosomal RNA function.

We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.

Base Sequence↗
PubMed2005

U2552 methylation at the ribosomal A-site is a negative modulator of translational accuracy.

We have recently identified RrmJ, the first encoded protein of the rrmJ-ftsH heat shock operon, as being the Um(2552) methyltransferase of 23S rRNA, and reported that rrmJ-deficient strains exhibit growth defects, reduced translation rates and reduced stability of 70S ribosomes. U2552 is an ubiquitously methylated residue. It belongs to the A loop of 23S RNA which is an essential component of the ribosome peptidyltransferase centre and interacts directly with aminoacyl(A)-site tRNA. In the present study, we show that a lack of U2552 methylation, obtained in rrmJ-deficient mutants, results in a decrease in programmed +1 and -1 translational frameshifing and a decrease in readthrough of UAA and UGA stop codons. The increased translational accuracy of rrmJ-deficient strains suggests that the interaction between aminoacyl-tRNA and U2552 is important for selection of the correct tRNA at the ribosomal A site, and supports the idea that translational accuracy in vivo is optimal rather than maximal, thus pointing to the participation of recoding events in the normal cell physiology.

Cell Cycle Proteins↗
PubMed1998

An extremely high conservation of RNA-protein S7 interactions during prokaryotic ribosomal biogenesis.

Direct determination of RNA-protein complex structures is often facilitated by the use of thermophilic proteins; however E. coli is the most investigated system so far. A hybrid approach is to form heterologous complexes of E. coli RNA with thermophilic proteins. The rationale for this approach to RNA-protein interactions in ribosomes is based on the ability of the thermophilic protein S7 to replace a homologous counterpart in vivo. In vitro, the protein S7 of Thermus thermophilus is able to form complexes with both the minimal 16S rRNA fragment and the intercistronic region of the str operon mRNA from E. coli (Kd = 1.4 x 10(7) M and 1.1 x 10(-7) M respectively). The interaction of Thermus S7 with the E. coli intercistronic mRNA is surprising, because this region does not exist in the thermophilic str operon. It suggests a high degree of conservation of an RNA-binding site on S7.

Bacterial Proteins↗
PubMed1990

Recent advances in peptide chain termination.

Peptide chain termination occurs when a stop codon is decoded by a release factor. In Escherichia coli two codon-specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required. The E. coli factors have been purified and their genes isolated. A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames. Frame-shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2-specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3' end of the 16S rRNA. The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map. Ribosomal binding studies place the RF-binding region at the interface between the ribosomal subunits. A possible mechanism of stop-codon recognition is reviewed.

Codon↗
PubMed1993

Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis.

Southern blot analysis of genomic DNA of the mesophilic lactic bacterium Lactococcus lactis subsp. lactis strain IL1403, illuminated six rRNA gene clusters. Each cluster contains one copy each of three rRNA genes, displaying the typical eubacterial organization of physically linked 16 S, 23 S and 5 S rRNA genes. Five of the six rRNA clusters were cloned into plasmid pBR322. One recombinant plasmid, pSLCM6, containing a 6500 base-pair genomic DNA fragment, was characterized by physical mapping and the sequences encoding rRNAs and tRNAs were localized by Southern hybridization. This fragment contains a single operon composed of one promoter, a leader sequence, a 16 S rRNA gene, a tRNA(Ala) gene, a 23 S rRNA gene, a 5 S rRNA gene and a tRNA(Asn) gene. S1 nuclease mapping and primer extension analysis of in vivo transcripts localized one transcriptional initiation site 150 base-pairs upstream from the start of the 16 S rRNA gene. These procedures also suggest that this transcript is processed by an RNAse III-like activity similar to Bacillus subtilis; i.e. the L. lactis nuclease might be sequence-specific. The chronology of specific cleavages occurring during the maturation process of the precursor transcript is described. One interesting observation is that the regions flanking the 16 S and 23 S rRNAs containing the primary processing sites are identical and contain sequences that could be involved in transcriptional antitermination. S1 mapping of the 3' ends of in vivo transcripts indicate that a terminator-like sequence a few base-pairs downstream from the distal tRNA(Asn) gene is inefficient in arresting transcription.

Base Sequence↗
PubMed2005

Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes.

Although it is well known that there is no long range colinearity in gene order in bacterial genomes, it is thought that there are several regions that are under strong structural constraints during evolution, in which gene order is extremely conserved. One such region is the str locus, containing the S10-spc-alpha operons. These operons contain genes coding for ribosomal proteins and for a number of housekeeping genes. We compared the organisation of these gene clusters in 111 sequenced prokaryotic genomes (99 bacterial and 12 archaeal genomes). We also compared the organisation to the phylogeny based on 16S ribosomal RNA gene sequences and the sequences of the ribosomal proteins L22, L16 and S14. Our data indicate that there is much variation in gene order and content in these gene clusters, both in bacterial as well as in archaeal genomes. Our data indicate that differential gene loss has occurred on multiple occasions during evolution. We also noted several discrepancies between phylogenetic trees based on 16S rRNA gene sequences and sequences of ribosomal proteins L16, L22 and S14, suggesting that horizontal gene transfer did play a significant role in the evolution of the S10-spc-alpha gene clusters.

Bacteria↗
PubMed1994

The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8-RNA interaction.

Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions.

Anions↗
PubMed1992

The role of rRNA bases in the interaction of peptidyltransferase inhibitors with bacterial ribosomes.

Synergism of streptogramins A (virginiamycin M, VM) and B (virginiamycin S, VS), peptidyltransferase inhibitors, was explored in EM4/pLC7-21 (wild type) and EM4/pERY (VS-resistant). These bacterial strains contained multicopy plasmids carrying an rrnH operon with wild type (pLC7-21) or mutated (A2058----U transversion) 23 S rRNA gene. Ribosomes with wild type and mutated rRNA were both present in EM4/pERY. The latter particles did not bind VS; in the presence of VM, however, high affinity VS binding occurred. As shown previously, VS protected against chemical reagents certain bases in domain V rRNA and VM in the stems flanking this loop. Differences between wild type and mutant ribosomes were observed: A2058, A2059, A2062, and G2505, protected by VS and ERY in EM4/pLC7-21, were unshielded in EM4/pERY. A2062 was shielded by VM in EM4/pERY, not in EM4/pLC7-21, and G2505 of mutant ribosomes became protected by VS when VM was simultaneously present. Induction by VM of a high affinity VS binding site in VS-sensitive and -resistant ribosomes indicates A2058 mutation to entail a conformational change of this site, which is counteracted by VM fixation. Accessibility of A2062 to chemical reagents (unlike behavior of EM4/pERY and EM4/pLC7-21 in the presence of VM) implies different conformations for wild type and mutant ribosomes.

Base Sequence↗
PubMed1983

Regions of DNA involved in the stringent control of plasmid-encoded rRNA in vivo.

We have examined the transcription of two plasmid-encoded, stable RNAs; a shortened 16S ribosomal RNA and the spacer transfer RNA2Glu from the Escherichia coli rrnB operon. Plasmid deletions were constructed in vitro, in order to examine the DNA regions required for stringent control of rRNA expression in vivo during amino acid starvation. We find that rRNA synthesized from plasmids does exhibit a relA-dependent, stringent response. The DNA sequences required for this regulation do not extend beyond 20 bases downstream of the P1 transcription initiation site. Deletion of P2, the second of the two tandem rRNA promoters, does not weaken the stringent control of transcripts from P1. These results demonstrate that pause sites for RNA polymerase identified in vitro do not play a significant role in the stringent control of rRNA synthesis in vivo and imply that stringent regulation takes place at the level of initiation, rather than elongation, of transcription. Surprisingly, we find that the presence of extra intact rrnB operons (carried by a multicopy plasmid) reduces the magnitude of the stringent response.

Amino Acids↗
PubMed1999

Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli.

The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end.

Bacterial Typing Techniques↗