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PubMed1988

Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein.

The spc ribosomal protein operon of Escherichia coli is feedback-regulated by ribosomal protein S8, a translational repressor. We have analyzed the region of the spc mRNA that is responsible for this regulation. First, we have established that the S8 target site on the mRNA is near the translation start site of the third gene encoding ribosomal protein L5 in the operon. This was done by constructing hybrid plasmids carrying spc operon ribosomal protein genes under lac transcriptional control, as well as their deletion derivatives, and carrying out both in vivo and in vitro protein synthesis experiments. Next, the secondary structure of this region was studied by analyzing 5' end-labeled RNA synthesized from the phage SP6 promoter using structure-specific nucleases. A secondary structure model consistent with the results was deduced with the aid of a computer prediction of RNA folding. In addition, we cloned and sequenced the corresponding region from Salmonella typhimurium, Proteus vulgaris and Serratia marcescens and found five "compensating" substitutions that support some of the deduced helical structures of mRNA. None of the base changes was inconsistent with the deduced secondary structure model. Finally, site-directed mutagenesis experiments have identified bases important for regulation, including two base-paired sites representing each of two helical regions. This has led to the conclusion that some specific nucleotide residues located between these two helical regions are directly involved in S8 recognition, and that the function of the two helical regions is to maintain the proper orientation of these nucleotide residues. Comparison of the structure of the S8 target site on the spc mRNA with the known S8 binding site on rRNA has revealed a striking similarity in both primary and secondary structures. In particular, primary sequences of rRNA conserved among distantly related bacterial species in this region is found to be identical with the sequences at the corresponding positions in mRNA. These results suggest that the same structural features of the S8 repressor protein are involved in the interaction with both 16 S rRNA and the mRNA target site.

Base Sequence↗
PubMed1996

Sequence variation in the putative replication origins of the five genetically distinct basic Euoenothera plastid chromosomes (plastomes).

Approximately 4,200 nucleotides of the 16S/23S rDNA spacer and the 5' region flanking the rrn operon of the plastid chromosomes representing the five basic, phylogenetically related Euoenothera plastomes were sequenced and compared. The sequences that harbor the putative replication origins are almost identical except for a 785-bp intercistronic segment between the genes for the 16S rRNA and trnI. Differences are mainly caused by insertions/deletions and duplications; the predicted potential for formation of quite extensive secondary structure differs among the plastomes. Unexpected intraplastome variation has also been noted. Furthermore, the sequence-based and published genetically deduced plastome pedigrees differ significantly.

Chromosomes↗
PubMed2004

Direct stimulation of the lambdapaQ promoter by the transcription effector guanosine-3',5'-(bis)pyrophosphate in a defined in vitro system.

The bacterial response to nutritional deprivation, called the stringent response, results in the introduction of the specific nucleotide guanosine-3',5'-(bis) pyrophosphate (ppGpp). This nucleotide interacts with RNA polymerase and alters its action so that transcription from certain promoters is inhibited, whereas transcription from others seems to be activated. The exact mechanism of transcriptional stimulation by ppGpp in vivo remains unknown. A passive control model has been proposed according to which transcription inhibition during the stringent response at several very active promoters, like those for rRNA and tRNA genes, makes more free RNA polymerase (RNAP) molecules available for transcription at promoters with weak binding affinities for RNAP, thus leading to their passive activation. Among promoters whose transcription is activated by ppGpp in vivo is the histidine operon promoter (hisGp). However, in vitro it is only possible to demonstrate this effect in a coupled transcription-translation system. Here we demonstrate, using another in vivo ppGpp-stimulated promoter, the phage lambdapaQ promoter, that activation by ppGpp in a defined in vitro system is direct. A systematic study of ppGpp effects on the stimulation of paQ revealed that, as in the case of promoters inhibited by this nucleotide, ppGpp decreases the half-life of paQ open complexes. Our results also indicate that the equilibrium binding affinity of RNA polymerase to paQ seems not to be affected in the presence of ppGpp. Our data indicate that the mechanism underlying ppGpp stimulation of paQ is due to an increased rate of productive open complex formation.

DNA-Directed RNA Polymerases↗
PubMed2003

Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.

Bacillus↗
PubMed1984

Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida.

The xylABC operon on the TOL plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylR. In the study here the nucleotide sequence was determined for the regulatory region of this operon. The in vivo transcription initiation site of the operon was determined by S1 nuclease and reverse transcriptase mapping. RNA was prepared from m-methylbenzyl alcohol-induced cells of Pseudomonas putida and Escherichia coli carrying pTN2, a derivative of the TOL plasmid containing the structural and regulatory genes of the entire toluene-degrading pathway. The amount of E. coli mRNA was estimated to be only 10% of that of P. putida mRNA. Consensus sequences of the -10 region (Pribnow box) and the -35 region (RNA polymerase recognition site) in E. coli genes were not found in the region preceding the transcription initiation site, whereas a sequence complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli existed in front of the predicted start codon of the xylA gene. These results explain the inefficient expression of TOL genes in E. coli.

Base Sequence↗
PubMed1992

Analysis of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions from Plesiomonas shigelloides.

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.

Base Sequence↗
PubMed1980

Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli.

The complete nucleotide sequence of the 23S RNA gene from the rrnB operon of Escherichia coli has been determined. The sequences of both strands of the entire gene were determined, most of the sequence was independently confirmed by use of alternate restriction fragments, and all restriction cuts overlapped. The DNA region corresponding to mature 23S rRNA contains 2904 nucleotides. Kethoxal-reactive sites protected by 30S subunits are found between positions 2300 and 28000, placing the subunit interface in this region of the molecule. The functional importance of this region is further supported by studies by other investigators, including homology with chloroplast and mitochondrial rRNA.

Base Sequence↗
PubMed1997

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

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

Arabidopsis↗
PubMed1983

Ribosomal RNA precursors of Bacillus subtilis.

The DNA sequence of the region corresponding to the 5'-end of a 16S rRNA gene of B. subtilis 168 was determined. Comparison of this sequence with the sequences flanking other 16S and 23S rRNA coding regions (1-4) indicated that large RNA stem structures, surrounding the mature 16S and 23S rRNAs, could form in a precursor rRNA. The 5'-ends of the precursors of 16S and 23S rRNAs (p16S and p23S) were mapped to the middles of these potential RNA stem structures. We propose that the initial cleavages of the primary rRNA transcript occur near the "opposed G's" which interrupt the basepairing of each of these stem structures. This model is supported by the finding that the 5'-end of the 5S rRNA precursor, p5A (5), maps to the region of the "opposed G's" in the 23S rRNA stem structure.

Bacillus subtilis↗
PubMed1993

Secondary structure and phylogeny of the chloroplast 23S rRNA gene from the brown alga Pylaiella littoralis.

The entire nucleotide sequence of a 23S rRNA gene from the brown alga Pylaiella littoralis (L.) Kjellm has been determined. The predicted length of the 23S rRNA is 2948 nucleotides, including the 4.5S rRNA-like region at the 3' end of the molecule. The putative transcript has been folded into a secondary structure by comparison to existing structure models, and the predicted helical regions were inspected by identifying compensatory downstream base changes. The 23S rRNA secondary structure presented here has features that are unique to P. littoralis (no other chromophyte or red algal 23S rRNA sequences are yet available), but has none of the features specific to the chloroplast rRNAs of green plants and green algae. The Pylaiella sequence was aligned with analogous plastidial and eubacterial gene sequences, and the alignment was used to construct a phylogenetic tree. The plastidial sequences formed a coherent cluster closely associated with the 23S rRNA of the cyanobacterium Anacystis nidulans. Within the plastid group, the P. littoralis sequence was most closely related to that of Euglena gracilis confirming earlier analyses based upon 16S rRNA sequences.

Base Sequence↗
PubMed1982

The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase/Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced amino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6-10 bp upstream from the initiation codon.

Amino Acid Sequence↗
PubMed2006

Molecular evolution of the nif gene cluster carrying nifI1 and nifI2 genes in the Gram-positive phototrophic bacterium Heliobacterium chlorum.

A major nif cluster was detected in the strictly anaerobic, Gram-positive phototrophic bacterium Heliobacterium chlorum. The cluster consisted of 11 genes arranged within a 10 kb region in the order nifI1, nifI2, nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB and nifV. The phylogenetic position of Hbt. chlorum was the same in the NifH, NifD, NifK, NifE and NifN trees; Hbt. chlorum formed a cluster with Desulfitobacterium hafniense, the closest neighbour of heliobacteria based on the 16S rRNA phylogeny, and two species of the genus Geobacter belonging to the Deltaproteobacteria. Two nifI genes, known to occur in the nif clusters of methanogenic archaea between nifH and nifD, were found upstream of the nifH gene of Hbt. chlorum. The organization of the nif operon and the phylogeny of individual and concatenated gene products showed that the Hbt. chlorum nif operon carrying nifI genes upstream of the nifH gene was an intermediate between the nif operon with nifI downstream of nifH (group II and III of the nitrogenase classification) and the nif operon lacking nifI (group I). Thus, the phylogenetic position of Hbt. chlorum nitrogenase may reflect an evolutionary stage of a divergence of the two nitrogenase groups, with group I consisting of the aerobic diazotrophs and group II consisting of strictly anaerobic prokaryotes.

Evolution, Molecular↗
PubMed1993

In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs.

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.

Base Sequence↗
PubMed1992

Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes.

Denitrification and methylotrophy in Paracoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced from P. denitrificans. A number of sequences are available for enzymes from Escherichia coli, Pseudomonas stutzeri and Pseudomonas aeruginosa. It is concluded that pathway specific c-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification. In methanol oxidation at least 20 genes are involved. In this case too pathway specific c-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholde-hydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. The moxFJGI operon determines the structural components of methanol dehydrogenase and the associated c-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The moxY protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy. The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA of Thiosphaera pantotropha is identical to that of P. denitrificans. Still these bacteria show a number of differences. T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the beta-and gamma-subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification in T. pantotropha, which belongs to the alpha-subdivision of purple non-sulfur bacteria is a remarkable property. Furthermore T. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochrome cd1 type as in P. denitrificans. Also the cytochrome b of the Qbc complex of T. pantotropha is highly similar to its counterpart in P. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the alpha-group of purple non sulphur bacteria is reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗
PubMed2000

Characterization of PaxA and its operon: a cohemolytic RTX toxin determinant from pathogenic Pasteurella aerogenes.

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

Abortion, Veterinary↗
PubMed1997

Decoding fidelity at the ribosomal A and P sites: influence of mutations in three different regions of the decoding domain in 16S rRNA.

The involvement of defined regions of Escherichia coli 16S rRNA in the fidelity of decoding has been examined by analyzing the effects of rRNA mutations on misreading errors at the ribosomal A and P sites. Mutations in the 1400-1500 region, the 530 loop and in the 1050/1200 region (helix 34) all caused readthrough of stop codons and frameshifting during elongation and stimulated initiation from non-AUG codons at the initiation of protein synthesis. These results indicate the involvement of all three regions of 16S rRNA in decoding functions at both the A and P sites. The functional similarity of all three mutant classes are consistent with close physical proximity of the 1400- 1500 region, the 530 loop and helix 34 and suggest that all three regions of rRNA comprise a decoding domain in the ribosome.

Amino Acid Sequence↗
PubMed2001

Rapid approach to determine rrn arrangement in Salmonella serovars.

A PCR method was developed by which to rapidly and accurately determine the rrn arrangement of Salmonella enterica serovars. Primers were designed to the genomic regions flanking each of the seven rrn operons. PCR analysis using combinations of these primers will distinguish each of the possible arrangements of the rrn skeleton.

Chromosome Mapping↗
PubMed1988

An archaebacterial promoter element for stable RNA genes with homology to the TATA box of higher eukaryotes.

The RNA polymerase of Methanococcus vannielii, in binary complex with two stable RNA operons, protects from exonuclease digestion the region from 32 bp upstream (-32) to 18 bp downstream (+18) of the transcription start site. Contained within this binding region, centered at -25, is an AT-rich sequence which is highly conserved upstream of 26 other archaebacterial tRNA and rRNA genes. We therefore propose the sequence TTTATAATA as a common element of promoters for stable RNA genes in archaebacteria. Both the similarity in sequence and the location of this conserved octanucleotide suggest homology to the eukaryotic TATA box preceding protein encoding genes transcribed by RNA polymerase B.

Base Sequence↗