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PubMed1996

Characterization of Rhizobium 'hedysari' by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting.

The taxonomic and discriminatory power of RFLP analysis of PCR amplified parts of rhizobial rrn operons was compared to those of genomic PCR fingerprinting with arbitrary and repetitive primers. For this purpose, the two methods were applied for characterization of a group of bacterial isolates referred to as Rhizobium 'hedysari'. As outgroups, representatives of the family Rhizobiaceae, belonging to the Rhizobium galegae, Rhizobium meliloti, Rhizobium leguminosarum and Agrobacterium tumefaciens species were used. By the RFLP analysis of the PCR products corresponding to the variable 5'-half of the 23S rRNA gene and of the amplified spacer region between the 16S and 23S rRNA genes all Rh. 'hedysari' strains studied were tightly clustered together while the outgroups were placed in an outer position. The PCR products of the 3' end parts of the 23S rDNA did not show significant RFL polymorphism and no species differentiation on their basis was possible. In parallel, analysis of the same strains was performed by PCR amplification of their DNA with 19, 18 and 10 bp long arbitrary primers (AP-PCR) as well as with single primers corresponding to several bacterial repetitive sequences (rep-PCR). By both AP and rep-PCR an identification of every particular strain was achieved. In general, all primers provided taxonomic results that are in agreement with the species and group assignments based on the RFLP analysis of the rrn operons. On the basis of the results presented here it can be concluded that AP and rep-PCR are more informative and discriminative than rDNA and RFLP analysis of the rhizobial strains studied.

Base Sequence↗

PubMed1987

Cloning and characterization of the 5' region of the cell wall protein gene operon in Bacillus brevis 47.

Bacillus brevis 47 secretes vast amounts of proteins derived from both middle wall protein (MWP) and outer wall protein into the medium. The 5' region of the cell wall protein gene operon was cloned into Bacillus subtilis and subsequently into B. brevis 47. On the basis of the nucleotide sequence analysis, an open reading frame coding for MWP was identified on the cloned DNA fragment. Two potential translation initiation sites for the MWP gene are located tandemly in the same reading frame. Each of the sites contains a sequence highly homologous to the 3' end of B. brevis rRNA and an initiation codon. The translational fusion of the 5' region of the MWP gene with the Bacillus licheniformis alpha-amylase gene resulted in the efficient expression of the alpha-amylase gene in B. brevis 47. Of the two potential translation initiation sites, the one located upstream could be eliminated without affecting the expression of the MWP-alpha-amylase fusion gene, suggesting that MWP is synthesized in a precursor form with a signal peptide of 23 amino acid residues. S1 nuclease mapping of the cell wall protein gene transcripts suggested the possibility of the existence of several promoters in the 5' region within 300 base pairs from the translation initiation sites; one promoter was definitely localized within this part of the 5' region, and it was capable of expressing a heterologous gene fusion at a high level. The roles of the apparent structural complexity of the 5' region of the cell wall protein gene operon are discussed in connection with the efficient gene expression.

Amino Acid Sequence↗

PubMed1996

The influence of base identity and base pairing on the function of the alpha-sarcin loop of 23S rRNA.

The alpha-sarcin loop of large subunit rRNAs is one of the sites of interaction of elongation factors with the ribosome, and the target of the cytotoxins alpha-sarcin and ricin. Using a genetic selection for increased frameshifting in a reporter gene, we have isolated a C --> U mutation at position 2666 in the alpha-sarcin loop. In the NMR-derived structure of the loop, bases equivalent to 2666 and 2654 are paired via a non-canonical base pairing interaction. Each of the three base substitutions at C2666 and A2654 was constructed by site-directed mutagenesis of a plasmid borne copy of the rrnB operon of Escherichia coli. Only the C2666 --> U and A2654 --> G mutations that resulted in the formation of canonical A-U and C-G base pairs respectively, increased the levels of stop codon readthrough and frameshifting. The effects of different base pair combinations at positions 2666 and 2654 on ribosome function were then tested by constructing and analyzing all possible base combinations at these sites. All A --> G base substitution mutations at position 2654 and C --> U substitutions at position 2666 increased the levels of translational errors. However, these effects were greatest when G2654 and U2666 had the potential to engage in standard Watson-Crick base pairing interactions. These data indicate that base identity as well as base pairing interactions are important for the function of this essential component of the large subunit rRNA.

Base Composition↗

PubMed2006

Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide.

Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA(Pro), the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNA(Pro) peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding/competition assays. The ribosome-TnaC-tRNA(Pro) complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNA(Pro) or its nonfunctional substitute, TnaC(W12R)-tRNA(Pro). Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNA(Pro) complexes but not of ribosome-TnaC(W12R)-tRNA(Pro) complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNA(Pro); it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNA(Pro) mutant complexes. These findings establish that Trp-12 of TnaC-tRNA(Pro) is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center.

Amino Acid Substitution↗

PubMed2001

Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling.

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha, beta, and gamma subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.

Bacteria↗

PubMed2002

Specific PCR amplification for the genus Pseudomonas targeting the 3' half of 16S rDNA and the whole 16S-23S rDNA spacer.

A PCR protocol was developed for the selective amplification of a segment of the ribosomal RNA operon in Pseudomonas strains. Two specific conserved sequences suitable for PCR priming were identified in the middle of the 16S rDNA and at the very beginning of the 23S rDNA respectively. As a result, amplified region includes the 3' half of the 16S rDNA with the whole 16S-23S rRNA Internal Transcripted Spacer (ITS1) sequence. The specificity of the primer set was checked on sequence databases and validated on collection strains and on one hundred soil bacterial isolates. Our results showed that both collection, soil-inhabiting Pseudomonas and some Pseudomonas-related Azotobacter DNAs could be amplified. This specific PCR for the detection of Pseudomonas strains was in good agreement with colony hybridisation using a Pseudomonas-specific probe. The targeted segment is relevant for a characterisation at the species (16S rDNA) as well as at the infraspecific (ITS1) levels. This PCR-based approach offers promising potential for the characterisation of environmental Pseudomonas populations.

3' Flanking Region↗

PubMed1986

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes.

In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors lambda EMBL3 and lambda EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.

Base Sequence↗

PubMed1983

Transcription initiation in eukaryotes: analysis of heterologous in vitro systems utilizing components from mammalian and yeast cells.

The properties of heterologous in vitro transcription systems utilizing components from mammalian and yeast cells have been investigated. Purified yeast RNA polymerase II, when supplemented with a full complement of mammalian transcription factors, does not promote specific transcription initiation on cloned mammalian class II genes. Similarly, a complete mammalian transcription system does not initiate specific transcription on cloned yeast class II genes. These results indicate evolutionary divergence in function of yeast and mammalian class II genes and the associated transcription apparatus. The functional differences observed in this study are corroborated by previously reported structural differences between yeast and mammalian RNA polymerase II and class II genes. In contrast, the mechanism of eukaryotic class III gene transcription appears to be evolutionarily conserved. Thus, a mammalian transcription extract specifically transcribes the cloned yeast 5S rRNA gene. This system synthesizes without processing the 130 base 5S rRNA precursor, and this primary transcript may be processed to the mature 120 base RNA using a partially purified yeast processing activity. An homologous yeast class III transcription system has also been developed. This yeast system contains all components necessary for proper synthesis, processing and splicing of yeast tRNA precursors. Using the homologous yeast transcription system, a template in which the 5' flanking region and first 5 base pairs of the 5S rRNA gene had been deleted is utilized to synthesize a 120 base transcript, but the efficiency of transcription is reduced to about 10% that of the wild type gene. Thus, the yeast 5S rRNA gene has an internal transcription control region, but the immediate 5' flanking sequence have effects on the level of transcription.

Animals↗

PubMed1990

Single base alterations upstream of the E. coli 16S rRNA coding region result in temperature-sensitive 16S rRNA expression.

We have isolated three new temperature-sensitive mutants of 16S rRNA, using the U1192 spectinomycin resistance as a selectable marker. These differ from our previously characterized ts mutants in that they map in the upstream leader region of the rRNA precursor (at positions -13, -30 and -59). The relative distribution of plasmid and chromosome-derived 16S rRNA is similar between 30S subunits, 70S ribosomes and polysomes at the permissive and restrictive temperatures. Processing of the 5' end of the RNA does not appear to be affected by the mutations. Second-site suppressors were found, and all of these except one (which is within 16S rRNA) were also due to point mutations in the upstream leader.

Base Sequence↗

PubMed1988

Arrangement of Dam methylation sites (GATC) in the Escherichia coli chromosome.

The occurrence of GATC (Dam-recognition) sites in available E. coli DNA sequences (representing about 2% of the chromosome) has been determined by a simple numerical analysis. Our approach was to analyze the nucleotide composition of nine large sequenced DNA stretches ("cantles") in order to identify patterns of GATC distribution and to rationalize such patterns in biological/structural terms. The following observations were made: (i) In addition to oriC, GATC-rich regions are present in numerous locations. (ii) There is a wide variation in GATC frequency both between and within DNA cantles which led to the identification of a void-cluster pattern of GATC arrangement. The distance between two GATCs was never greater than 2 kb. (iii) GATC sites are found more frequently in translated regions than (in decreasing order) non-coding or non-translated regions. In particular, rRNA and tRNA encoding genes exhibit the lowest GATC content.

Base Composition↗

PubMed1994

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.

Base Sequence↗

PubMed1999

RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria.

A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity and extreme thermostability of purified A. pyrophilus RNA polymerase were verified. Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide. Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (beta and beta') showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A. pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits beta and beta', A. pyrophilus was placed within the Gram-negative bacteria below the epsilon subdivision of the Proteobacteria. No support was found for the 16S rRNA-based hypothesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies. This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms.

Adenosine Triphosphatases↗

PubMed1983

Plasmid transformation in Bacillus subtilis. Effects of the insertion of Bacillus subtilis rRNA genes into plasmids.

Two HindIII generated DNA fragments of 3.0 and 2.3 Kb derived from rRNA genes of B. subtilis were cloned in E. coli with pBR322. The 3.0 Kb fragment could be subcloned in B. subtilis using pC194. However, only the multimeric, but not the monomeric derivatives of this hybrid plasmid were active in transformation of B. subtilis cells. The 2.3 Kb fragment could neither be subcloned in pC194 nor in pPL603, using both cell and protoplast transformation. We attribute the nonclonability of the 2.3 Kb fragment in B. subtilis to the presence of strong promoter activity in this fragment. Direct proof for the presence of a strong promoter, which is apparently responsible for the transcription of the rRNA gene, was obtained in experiments with B. subtilis and E. coli promoter search plasmids.

Bacillus subtilis↗

PubMed2001

Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA.

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF-lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem-loop with that of Salmonella enterica serovar Typhimurium or a stem-loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon-rRNA base pairing does not occur.

Bacterial Proteins↗

PubMed1995

A polymerase switch in the synthesis of rRNA in Saccharomyces cerevisiae.

Transcription of ribosomal DNA by RNA polymerase I is believed to be the sole source of the 25S, 18S, and 5.8S rRNAs in wild-type cells of Saccharomyces cerevisiae. Here we present evidence for a switch from RNA polymerase I to RNA polymerase II in the synthesis of a substantial fraction of those rRNAs in respiratory-deficient (petite) cells. The templates for the RNA polymerase II transcripts are largely, if not exclusively, episomal copies of ribosomal DNA arising from homologous recombination events within the ribosomal DNA repeat on chromosome XII. Ribosomal DNA contains a cryptic RNA polymerase II promoter that is activated in petites; it overlaps the RNA polymerase I promoter and produces a transcript equivalent to the 35S precursor rRNA made by RNA polymerase I. Yeast cells that lack RNA polymerase I activity, because of a disruption of the RPA135 gene that encodes subunit II of the enzyme, can survive by using the RNA polymerase II promoter in ribosomal DNA to direct the synthesis of the 35S rRNA precursor. This polymerase switch could provide cells with a mechanism to synthesize rRNA independent of the controls of RNA polymerase I transcription.

Base Sequence↗

PubMed1996

Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts.

Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA-lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5'-untranslated region of the mRNA are required for translation. Two of them are complementary to the 3'-terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans-acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3'-end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans-acting factor(s).

Base Sequence↗

PubMed2005

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic.

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.

Czech Republic↗

PubMed2002

Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way.

5' Untranslated Regions↗