Complete nucleotide sequence of the 26S rRNA gene of Physarum polycephalum: its significance in gene evolution.

The complete nucleotide sequences of the 5.8S and 26S rRNA genes of Physarum polycephalum and the transcribed spacer between them were determined. Comparison of the sequences with those of the Escherichia coli 23S rRNA and yeast 26S rRNA genes showed that there are 16 highly homologous regions in the sequences of Physarum and E. coli and that eukaryotes have some eukaryote-specific extra sequences. The sequence immediately following the 5.8S-like region of E. coli 23S rRNA was found to be highly homologous to the 5' terminus of Physarum 26S rRNA, indicating that the eukaryote-specific 5.8S rRNA gene is derived from the 5'-terminal region of the prokaryote large rRNA gene.     

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.     

鼠疫耶尔森氏菌16S-23SrRNA基因间区分析

目的　发展一种快速准确的鉴定鼠疫菌的方法。方法　 Ｐ Ｃ Ｒ 反应加限制性内切酶消化的方法。结果　对来自不同疫源地的 １０３ 株鼠疫菌 １６ Ｓ２３ Ｓｒ Ｒ Ｎ Ａ 基因间区进行扩增,均可扩出两条长为 １ １４６ｂｐ 及 １ ０９０ｂｐ 的片段,扩增产物用限制性内切酶 Ｔａｑ Ｉ, Ｍ ｓｐ Ｉ消化后的酶切图谱相同。而对照菌株小肠结肠炎耶尔森氏菌,鼠伤寒沙门氏菌,大肠杆菌,痢疾杆菌,霍乱弧菌的扩增产物及酶切图谱与鼠疫菌均不相同。结论　鼠疫菌 １６ Ｓ２３ Ｓｒ Ｒ Ｎ Ａ 基因间区高度同源,基本为两种类型,长度分别为 １ １４６ｂｐ 及 １ ０９０ｂｐ,其他菌株与其明显不同;该方法将有助于快速准确的鉴定鼠疫菌。     

Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.     

Synthesis of a large precursor to ribosomal RNA in a mutant of Escherichia coli

A mutant of E. coli, isolated by Kindler and Hofschneider as a strain defective in RNase III activity, forms a 30S precursor of ribosomal RNA ("30S pre-rRNA"). The half-life of the 30S pre-rRNA in growing cells at 30 degrees , estimated by the rate of specific (3)[H]uridine incorporation, is about 1 min. In rifampicin-treated cells, the RNA is metabolized to mature rRNA with a half-life of about 2 min.The 30S pre-rRNA has been highly purified. DNA-RNA hybridization tests demonstrate that it contains both 16S and 23S rRNA sequences. Also, in cultures treated with rifampicin, the cleavage products of radioactive 30S pre-rRNA include 25S and 17.5S RNA species, destined to becomes 23S and 16S rRNA. Thus, each 30S chain probably contains one 16S and one 23S RNA sequence, as well as additional sequences. Two independent techniques indicate that the additional portions account for about 27% of the total lenght: (1) By comparison to the sedimentation rate and electrophoretic mobility of marker RNAs, the 30S pre-RNA has an apparent molecular weight of 2.3 x 10(6) +/- 5%, or 28% more than the sum of 16S and 23S rRNA; (2) 27% of the 30S pre-rRNA is not competed away from hybridization by mature 16S and 23S rRNA.Thus, bacteria appear to make a pre-rRNA similar in some respects to that observed in eukaryotes; though in normal E. coli cells, the pre-rRNA is ordinarily cleaved endonucleolytically during its formation.     

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.     

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.     

Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes.

We have constructed a conditional rRNA gene expression system by fusing a plasmid-encoded rrnB operon to the lambda PL promoter/operator. It was thereby possible to study the events that lead to the regulation of chromosomal rRNA and tRNA synthesis after overproduction of rRNA. rRNA induction resulted in a 2-fold increase in 30S and 50S free ribosomal subunits, whereas the polysome fraction was unaffected. Overproduction of rRNA and "free" ribosomes produced a large repression of rRNA and tRNA synthesis from chromosomal genes and a smaller increase in the concentration of guanosine tetraphosphate. These results lend support to the ribosome feedback regulation model: rRNA and tRNA operons are negatively regulated, either directly or through some intermediate, by free, nontranslating ribosomes.     

Identification of spacer tRNA genes in individual ribosomal RNA transcription units of Escherichia coli.

Transfer RNA genes ("spacer tRNA genes") are present in the spacer region between 16S and 23S rRNA genes in Escherichia coli. We have analyzed spacer tRNA genes carried by seven rRNA operons with different chromosomal locations. Six of these were isolated on plasmids and one on a transducing phage. We found that, in addition to the two previously identified genes for tRNA2Glu and tRNAIIle, there is a spacer tRNA gene which codes for tRNAIBAla. Of the seven rRNA operons studied, three had both tRNAIBAla and tRNAIIle genes, and the remaining four had the tRNA2Glu gene in their spacers. In addition, genes for tRNAIAsp were found near the distal ends of two different rRNA operons.     

Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria

The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis, regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition-fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes.     

Specific binding of tRNAMet to 23S rRNA of Escherichia coli.

tRNAMetf binds to 23S rRNA of Escherichia coli, forming a complex with a melting temperature of 75 degrees (in 0.6 M NaCl). The regions within the RNAs that bind to each other have been isolated and their nucleotide sequences have been determined. The interacting region in tRNAMetf is 17 nucleotides long, extending from G5 in the acceptor stem to D21 (D = 5.6-dihydrouridine) in the D loop. The sequence in 23S rRNA is complementary to that sequence except for an extra Up in the middle and allowing a Gp.D base pair. We propose that association of these two sequences may play a role in initiation of protein synthesis by tRNAMetf. In addition, part of this sequence in 23S rRNA may also stabilize tRNA binding to the ribosome during elongation of nascent polypeptides.     

Isolation and identification of Escherichia albertii from a patient in an outbreak of gastroenteritis

A microbial strain harboring the eae gene, which is known as the virulence gene of enteropathogenic Escherichia coli (EPEC) and most enterohemorrhagic E. coli, was isolated from a patient in a gastroenteritis outbreak that occurred in 22 patients in Akita Prefecture, Japan, in November 2011. The biochemical characteristics of the isolate were more similar to those of a novel Escherichia sp., E. albertii than E. coli. Partial 16S rRNA gene sequences of the isolate were identical to those of a certain E. albertii strain, but also showed a high degree of similarity to those of E. coli strains. Finally, we identified this isolate as E. albertii by performing PCR analysis that targeted the uidA, lysP, mdh, and cdtB genes in addition to stx and eae genes to differentiate between the EPEC and E. albertii strains.     

Processing of the 5'' end of Escherichia coli 16S ribosomal RNA.

We have isolated and partially characterized an endonuclease involved in processing the 5' end of 16S rRNA of Escherichia coli. A mutant strain that is deficient in this enzyme accumulates a new precursor of 16S rRNA, named 16.3S rRNA. This rRNA has the 3' end of mature 16S rRNA but is about 60 nucleotides longer at the 5' end. In vitro, the enzyme preparation cleaves an RNA fragment of about 60 nucleotides from the 5' end of 16.3S rRNA in 30S ribosomal subunits, yielding the mature 5' end of 16S rRNA. In the mutant strain the 16.3S rRNA is associated with a full complement of 21 ribosomal proteins in 30S subunits. These particles, which comprise 50% of the total 30S subunits, are present on polyribosomes.     

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.     

立克次体的16SrRNA基因序列分析

目的　研究立克次体的１６ＳｒＲＮＡ基因序列的差异,为其分类和鉴定提供依据。方法　从基因数据库中选择不同的立克次体属代表种的１６ＳｒＲＮＡ基因序列,在计算机上用多序列排列程序对序列进行同源位排列和对比分析,以及依据序列的相似程度构建进化树。结果　立克次体的１６ＳｒＲＮＡ基因含有４ 个高变区,这些高变区的序列具有种或属的特异性,依据序列的相似程度构成的进化树可将立克次体目内成员分为柯克斯体、巴通体、立克次体、埃立克体等４ 个大基因群,每个大基因群含有若干个小基因群。结论　１６ＳｒＲＮＡ基因高变区的序列可用于设计立克次体种、属特异性ＰＣＲ引物和杂交探针;１６ＳｒＲＮＡ 基因序列的分析是目前立克次体分类和鉴定的最可靠依据