Electron microscopic analysis of the transcription of the Bacillus subtilis riboflavin operon inserted into the hybrid plasmid pLP102 (original) (raw)
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Riboflavin operon of Bacillus subtilis : unusual symmetric arrangement of the regulatory region
Molecular and General Genetics, 1992
Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides + 37 and + 159 relative to the transcription initiation site of the riboflavin operon. This region displays an unusual structure for regulatory sequences. The main part of it represents clusters of A/T and G/Grich sequences that symmetrically blank a short inverted repeat.
Microbiology-sgm, 1999
A 3-5 kb EcoRl-BamHI fragment of Baci//us subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-€scherichia coli shuttle vector pCB2O. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of microorganisms , such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of f lavokinase activity in cell extracts demonstrated that ribR encodes a monof unctional f lavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.
Nucleic Acids Research, 1989
A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned £. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the fi. subtilis cloned DNA indicated a high degree of conservation of SPC operon ribosomal protein genes between fi. subtilis and £. coli. This fragment contains DNA homologous to the promoter-proximal region of the SPC operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B_. subtilis genes in this region is identical to that found in £. coli. A region homologous to the £. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the SPC operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with £• coli sequences, we failed to find sequences which would form a structure resembling the £. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in £• coli. in this region or elsewhere in the B. subtilis BPC DNA.
The Journal of General and Applied Microbiology, 2006
Most bacteria have multiple rrn operons and encode rRNA genes, and the majority of RNA synthesized at rapid growth rates is rRNA, probably at least in part due to its copy number (Bremer and Dennis, 1996; Jensen and Pedersen, 1990; Lewis et al., 2000). The complete DNA sequence of Bacillus subtilis 168 genome contains 10 rRNA operons (Kunst et al., 1997). Each operon encodes for three rRNA molecules; 16S, 23S and 5S rRNA. [There are two obvious s A consensus promoter sequences (P1 and P2) in the transcriptional regulatory regions of the rrn operons (Ogasawara et al., 1983; Stewart and Kenneth, 1983; Wellington and Spiegelman, 1993) except for the rrnE operon with three promoters (P1, P2 and P3) (Fig. 1, Kawamura, F. unpubl.).] Several clusters of tRNA genes are found downstream of 5S rRNA genes (for rrnJ, I, E, D and B), in the spacer regions between the 16S rRNA and 23S rRNA genes (for rrnO, rrnA), and upstream of 16S rRNA gene (for rrnE). Moreover, rrnJ-W and rrnI-H-G are contiguous operons. It has also been reported that some laboratory strains of B. subtilis contain only 9 rrn operons due to the spontaneous deletions occurring within these clusters (Widom et al., 1998). We therefore focused on the 7 rrn operons, including the first operons of contiguous clusters, rrnJ and I, and the noncontiguous operons, rrnO, A, E, D and B (Fig. 1). It should be useful for monitoring transcription activity of each rrn operon in B. subtilis to construct transcriptional bgaB fusions with each regulatory region of transcription in the rrn operons at the amyE locus. The transcriptional regulatory regions including P1 and P2 promoters of rrnO, A, J, I, D or B, as well as P1, P2 and P3 promoters of rrnE operon in B. subtilis 168 DNA were PCR-amplified by use of primers listed in Table1. The forward primers, OF , A-F, J-F, IF , E-F, D-F and B-F, were designed in the upstream genes of each operon and the common reverse primers, rrn-R1 for rrnO, E, D and B and rrn-R2 for rrnA, I and J, were designed immediately upstream of the mature 16S rRNA. The leader sequences, from the putative initiation site of transcription through the 5Ј-terminal nucleotide of the 16S rRNA gene, are highly conserved.
European journal of biochemistry, 1996
The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced. The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 21 3 amino acids with a m i s s of 23.4 kDa. It was mapped to a position at 37.5 min on the physical map of the E. coli chromosome. The 3' end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase. This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively. The gene ydhhc is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein. The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein. The ydhB gene is directly adjacent to the regulatory gene purR. A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min. The ribC gene was hyperexpressed in recombinant E. coli strains to a level of about 30 o/c of cellular protein. The protein was purified to homogeneity by chromatography. The specific activity was 26000 nmol. mg. h-'. The protein sediments at a velocity of. F~~) = 3.8 S. Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure. The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the Cterminal and N-terminal parts) suggesting two structurally similar folding domains.
Journal of Molecular Biology, 1970
A procedure for the purification of specific genes ae DNA-RNA hybrids is described. The ribosomal RNA genee of Badha mbtilia were selected as a model system for this study, since the ribosomal RNA from this species can be readily obtained in pure form and since much biochemical and genetic background information about the genes was already available. B. m.btiZir, DNA was mechanically sheared and the complementary strands were separated by methylated albumin kieaelguhr column chromatography. The ribosomal RNA gene fraction was then identified by hybridization with a mixture of 16 s and 23 s ribosomal RNA. Passage through a hydroxyapatite column eliminated any renatured DNA. The DNA-RNA hybrid was then separated from the bulk, nonhybridized, single-stranded DNA by ceaium sulfate den&y-gradient centrifugation in the presence of mercuric ions. The overall purification was SO-fold, aa measured by the increase in hybridizing ctctivity with 16 s or 23 s ribosomal RNA. This result indicated that at least 30% of the purified DNA was composed of ribosomal RNA genes. The techniques used for the purification are discussed in detail and future applications are considered.
Chromosomal organization of rRNA operons in Bacillus subtilis
Genetics, 1988
Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion.
MGG Molecular & General Genetics, 1987
Sequencing data indicated that the RNA polymerase o'43 operon of Bacillus subtilis consisted of three genes, P23 (function unknown), dnaE (DNA primase), and rpoD (0-43) (Wang and Doi 1986a). S1 nuclease mapping experiments with RNA from various stages of growth demonstrated the presence of two overlapping 0-43 promoters that controlled the expression of the operon during growth and a 0 -37 promoter that regulated the expression of the operon during the sporulation phase. This promoter switching mechanism ensured that this important operon would be expressed during different nutritional states of the cell and also illustrated a function for the minor RNA polymerase 0-37 holoenzyme in the expression of genes which are normally expressed during the logarithmic phase of growth. The location of the transcription termination signal confirmed that the o'43 operon consists of three genes.