Asymmetric template function of microbial deoxyribonucleic acids: transcription of ribosomal and soluble ribonucleic acids

In Bacillus subtilis and Escherichia coli, 16 and 23S ribosomal ribonucleic acid (rRNA) hybridize exclusively with the heavy (H) strand of methylated albuminkieselguhr (MAK)-fractionated complementary deoxyribonucleic acid (DNA) strands. All the soluble RNA (4S RNA) in B. subtilis and 66 to 75% of the 4S RNA in E. coli also hybridize with the H strand. Interspecific hybridization shows that E. coli 23S rRNA also binds selectively to the DNA H strand of Salmonella typhimurium. The hybridization peak for all three cellular RNA components is specifically located in the late-eluting region of the absorbance profile of the DNA H strand. The early-eluting region of the light (L) strand preferentially inhibits the hybridization between the peak region of the H strand and 23S rRNA. These regions are considered to represent the transcribing sequences and their complements for 23S rRNA in the separated H and L strands of DNA, respectively.     

tRNA genes are found between 16S and 23S rRNA genes in Bacillus subtilis.

There are at least nine, and probably ten, ribosomal RNA gene sets in the genome of Bacillus subtilis. Each gene set contains sequences complementary to 16S, 23S and 5S rRNAs. We have determined the nucleotide sequences of two DNA fragments which each contain 165 base pairs of the 16S rRNA gene, 191 base pairs of the 23S rRNA gene, and the spacer region between them. The smaller space region is 164 base pairs in length and the larger one includes an additional 180 base pairs. The extra nucleotides could be transcribed in tRNAIIe and tRNA Ala sequences. Evidence is also presented for the existence of a second spacer region which also contains tRNAIIe and tRNA Ala sequences. No other tRNAs appear to be encoded in the spacer regions between the 16S and 23S rRNA genes. Whereas the nucleotide sequences corresponding to the 16S rRNA, 23S rRNA and the spacer tRNAs are very similar to those of E. coli, the sequences between these structural genes are very different.     

Conserved portion in bacterial ribosomal RNA

The conserved portion in bacterial ribosomal RNA was studied by the DNA-RNA hybridization method. The hybridization percentages were as follows: Bacillus subtilis DNA and B. subtilis 23S rRNA, 0.16; Escherichia coli DNA and E. coli 23S rRNA, 0.15; B. subtilis DNA and E. coli 23S rRNA, 0.03; E. coli DNA and B. subtilis 23S rRNA, 0.04. The RNA's extracted from the heterologous hybrids could be rehybridized with DNA's of B. subtilis and E. coli. The average chain lengths of the RNA's were estimated by sucrose density gradient centrifugation and Sephadex gel filtration. The results suggested that the size might be larger than 30 nucleotides. Nucleotide compositions of the RNA's in the hybrids were also studied. Both RNA's contained higher molar percentages of guanylic acid and cytidylic acid than the whole rRNA's.     

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.     

Sequence of the gene for isoleucine tRNA1 and the surrounding region in a ribosomal RNA operon of Escherichia coli

A DNA fragment of about 2000 base pairs carrying the gene for tRNA(1) (Ile) has been cloned from a total Eco RI endonuclease digest of Escherichia coli DNA. Sequence analyses revealed that about the first 850 base pairs from one end of the fragment contain a nucleotide sequence corresponding to that in the 3'-end of 16S rRNA. The gene for tRNA(Ile) follows the 16S rRNA gene and both genes flank a spacer sequence of 68 base pairs. The spacer region contains a repeating, a hair pin and a symmetrical structure when the sequence is viewed in the single stranded form. A notable hair pin structure is also observed in the region adjacent to the 3'-end of the tRNA(1) (Ile) gene. In addition, about 850 base pairs from the other end of the DNA fragment have been found to contain the nucleotide sequence of the 5'-end of 23S rRNA. The presence of the genes for tRNA(1) (Ile), 16S and 23S rRNA and the hybridization to tRNA(1) (Ala) suggest that this cloned DNA is part of one of the E. coli rRNA operons carrying these two tRNA genes as a spacer.Images     

Characterization of Paenibacillus popilliae rRNA operons

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.     

Sequence of a putative promoter region for the rRNA genes of tobacco chloroplast DNA.

The nucleotide sequence of the segment of tobacco chloroplast DNA adjacent to and including the start of the 16S rRNA gene has been determined. The region just preceding this gene was found to contain a tRNAVal gene and promoter-type sequences similar to those which occur in E. coli were found before this tRNA gene. E. coli RNA polymerase can recognize these sequences and in vitro co-transcribes the tRNA and rRNA genes.     

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.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.     

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.