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

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and 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.     

In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit

Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a “folding competent” state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation.     

Prediction of secondary structures of 16S and 23S rRNA fromEscherichia coli

Small and large subunits ofEscherichia coli ribosome have three different rRNAs, the sequences of which are known. However, attempts by three groups to predict secondary structures of 16S and 23S rRNAs have certain common limitations namely, these structures are predicted assuming no interactions among various domains of the molecule and only 40% residues are involved in base pairing as against the experimental observation of 60 % residues in base paired state. Recent experimental studies have shown that there is a specific interaction between naked 16S and 23S rRNA molecules. This is significant because we have observed that the regions (oligonucleotides of length 9–10 residues), in 16S rRNA which are complementary to those in 23S rRNA do not have internal complementary sequences. Therefore, we have developed a simple graph theoretical approach to predict secondary structures of 16S and 23S rRNAs. Our method for model building not only uses complete sequence of 16S or 23S rRNA molecule along with other experimental observations but also takes into account the observation that specific recognition is possible through the complementary sequences between 16S and 23S rRNA molecules and, therefore, these parts of the molecules are not used for internal base pairing. The method used to predict secondary structures is discussed. A typical secondary structure of the complex between 16S and 23S rRNA molecules, obtained using our method, is presented and compared Briefly with earlier model Building studies.     

Methylated 23S rRNA nucleotide m2G1835 of Escherichia coli ribosome facilitates subunit association

Among 4.5 thousand nucleotides of Escherichia coli ribosome 36 are modified. These nucleotides are clustered in the functional centers of ribosome, particularly on the interface of large and small subunits. Nucleotide m²G1835 located on the 50S side of intersubunit bridge cluster B2 is modified by N2-methyltransferase RlmG. By means of isothermal titration calorimetry and Rayleigh light scattering, we have found that methylation of m²G1835 specifically enhances association of ribosomal subunits. No defects in fidelity of translation or interaction with translation GTPases could be ascribed to the ribosomes unmethylated at G1835 of the 23S rRNA. Methylation of G1835 was found to provide a significant advantage for bacteria at osmotic and oxidative stress.     

大肠杆菌16S rRNA的核酶设计和初步应用

针对细菌rRNA研发抑制细菌增殖的新型抗菌素是抗生素研究领域的新课题。细菌rRNA与基因mRNA一样自然形成折叠卷曲高级结构,其结构上可以结合反义核酸的位点即靶点,靶点的阐明是设计有效反义核酸、核酶（Ribozyme）和脱氧核酶（DNAzyme）的关键。MAST方法固定16S rRNA,将其与寡核苷酸文库杂交筛选出靶点,获得了大肠杆菌16S rRNA的6个反义核酸结合靶点,并鉴定5个靶点有效,其中1个为高效。5个靶点的反义核酸能在通透性大肠杆菌菌株培养中不同程度地抑制其生长,针对高效靶点的核酶在转化大肠杆菌中表达而抑制其生长。     

Identification of 5-hydroxycytidine at position 2501 concludes characterization of modified nucleotides in E. coli 23S rRNA

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA—but a single modified nucleoside at position 2501 in 23S rRNA—has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine—a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.     

The rRNA operon from Zea mays chloroplasts: nucleotide sequence of 23S rDNA and its homology with E.coli 23S rDNA.

The nucleotide sequence of 23S rDNA from Zea mays chloroplasts has been determined. Alignment with 23S rDNA from E.coli reveals 71 percent homology when maize 4.5S rDNA is included as an equivalent of the 3' end of E.coli 23S rDNA. Among the conserved sequences are sites for base modification. Chloramphenicol sensitivity and ribosomal subunit interaction. A proposal for the base pairs formed between 16S and 23S rRNAs during the 30S/50S subunit interaction is presented. The alignment of maize 23S rDNA with that of E.coli reveals three small insertion sequences of 25, 65 and 78 base pairs, whereas maize 16S rDNA shows only deletions when compared with the E.coli species.     

Using protein synthesis inhibitors to establish the phylogenetic relationships of the sulfolobales order

The sensitivity of the cell-free protein synthesis systems from Acidanus brierleyi, Acidianus infernus, and Metallosphaera sedula, members of the archaeal order Sulfolobales, to 40 antibiotics with different specificities has been studied. The sensitivity patterns were compared to those of Sulfolobus solfataricus and other archaeal, bacterial, and eukaryotic systems. The comparative analysis shows that ribosomes from the sulfolobales are the most refractory to inhibitors of protein synthesis described so far. The sensitivity results have been used to ascertain in phylogenetic relationships among the members of the order Sulfolobales. The evolutionary significance of these results are analyzed in the context of the phylogenetic position of this group of extreme thermophilic microorganisms. Correspondence to: R. Amils     

Chloramphenicol-erythromycin resistance mutations in a 23S rRNA gene of Escherichia coli.

Two chloramphenicol resistance mutations were isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Both mutations also confer resistance to 14-atom lactone ring macrolide antibiotics, but they do not confer resistance to 16-atom lactone ring macrolide antibiotics or other inhibitors of the large ribosomal subunit. Classic genetic and recombinant DNA methods were used to map the two mutations to 154-base-pair regions of the 23S RNA genes. DNA sequencing of these regions revealed that chloramphenicol-erythromycin resistance results from a guanine-to-adenine transition at position 2057 of the 23S RNA genes of both independently isolated mutants. These mutations affect a region of 23S RNA strongly implicated in peptidyl transfer and known to interact with a variety of peptidyl transferase inhibitors.     

Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli

4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA.     

Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin.

Summary Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.     

Methylation at nucleotide G745 or G748 in 23S rRNA distinguishes Gram-negative from Gram-positive bacteria

Bacteria tune the function of their ribosomes by methylating specific rRNA nucleotides. Nucleotide G745 in Escherichia coli 23S rRNA is methylated by the methyltransferase enzyme RrmA, whereas in Streptomyces fradiae, the neighbouring nucleotide G748 is methylated by the enzyme TlrB. Both nucleotides line the peptide exit channel of the ribosome at the binding site of macrolide, lincosamide and streptogramin B antibiotics. Resistance to the macrolide tylosin, which is produced by S. fradiae, is conferred by methylation of G748. RrmA and TlrB are homologues (29% identical), and a database search against all presently available sequences revealed a further two dozen homologues from a wide variety of Bacteria. No homologues were found among the Archaea or Eukarya. The bacterial sequences adhere to the species phylogeny and segregate into two groups, in which the Gram-negative sequences align with RrmA and the Gram-positives with TlrB. Consistently, in more than 20 species tested, the distribution of methylation in the Gram-negative rRNAs (methylated at G745) and the Gram-positives (methylated at G748) perfectly matches the bacterial phylogeny. Cloning and expression of representative methyltransferase genes showed that this specificity of methylation is determined solely by the methyltransferase enzyme and is independent of the origin of the rRNA substrate. This is the first case in which the position of an RNA methylation defines a sharp division between the Gram-negative and Gram-positive bacteria. Given the specificities and distribution of these methyltransferases, we propose a change in the nomenclature of RrmA to RlmAI (rRNA large subunit methyltransferase) and of TlrB to RlmAII.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

Ribosome origins: The relative age of 23S rRNA Domains

The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.     

Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy.

We have produced mutations in a cloned Escherichia coli 23S rRNA gene at positions G2252 and G2253. These sites are protected in chemical footprinting studies by the 3' terminal CCA of P site-bound tRNA. Three possible base changes were introduced at each position and the mutations produced a range of effects on growth rate and translational accuracy. Growth of cells bearing mutations at 2252 was severely compromised while the only mutation at 2253 causing a marked reduction in growth rate was a G to C transversion. Most of the mutations affected translational accuracy, causing increased readthrough of UGA, UAG and UAA nonsense mutations as well as +1 and -1 frameshifting in a lacZ reporter gene in vivo. C2253 was shown to act as a suppressor of a UGA nonsense mutation at codon 243 of the trpA gene. The C2253 mutation was also found not to interact with alleles of rpsL coding for restrictive forms of ribosomal protein S12. These results provide further evidence that nucleotides localized to the P site in the 50S ribosomal subunit influence the accuracy of decoding in the ribosomal A site.     

YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA

Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Ψ) sites in Escherichia coli rRNA, and further modification is found only at Ψ1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specifically catalyzes methyl group addition to form m³Ψ1915. Analyses of E. coli rRNAs using MALDI mass spectrometry showed that inactivation of the ybeA gene leads to loss of methylation at nucleotide Ψ1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy of ybeA. Homologs of the ybeA gene, and thus presumably the ensuing methylation at nucleotide m³Ψ1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss of ybeA function in E. coli causes a slight slowing of the growth rate. Phylogenetically, ybeA and its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576; ybeA is the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Ψ1915. Methylation by YbeA (redesignated RlmH for rRNA large subunit methyltransferase H) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation.