rRNA topography in ribosome. IV. The accessibility of the 5'-end region of 16S rRNA

The accessibility of the 5'-end region of 16S rRNA (A₈GAGUUUG₁₅) inEscherichia coli ribosomes for complementary binding with the synthetic octanucleotide d(CAAACTCT) has been studied. Nonequilibrium gel-filtration was used to evaluate parameters of the binding of this oligonucleotide with free 16S rRNA, ribosomal subunits, and 70S ribosomes. A simple approach is presented to calculate the apparent association constants and the number of binding sites based upon the data obtained under those conditions. Free 16S rRNA, 30S subunits, and 70S ribosomes were found to form rather stable complexes with the octanucleotide, the association constants being similar in all three cases. These data strongly suggest the surface location of the 16S rRNA 5'-end inE. coli ribosomes.     

Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis.

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with large internal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and 23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNA synthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA and defective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did not result in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16S RNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. Defective RNA species also may be degraded for this reason or because proper ribosome assembly is prevented by the defects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduction of binding sites for feedback ribosomal protein does not result in significant unbalanced translational feedback depression of ribosomal protein mRNAs.     

5S rRNA Data Bank.

In this paper we present the updated version of the compilation of 5S rRNA and 5S rDNA nucleotide sequences. It contains 1622 primary structures of 5S rRNAs and 5S rRNA genes from 888 species. These include 58 archaeal, 427 eubacterial, 34 plastid, nine mitochondrial and 1094 eukaryotic DNA or RNA nucleotide sequences. The sequence entries are divided according to the taxonomic position of the organisms. All individual sequences deposited in the 5S rRNA Database can be retrieved using the WWW-based, taxonomic browser at http://rose.man.poznan.pl/5SData/5SRNA.html++ + or http://www.chemie. fu-berlin.de/fb_chemie/agerdmann/5S_rRNA.html . The files with complete sets of data as well as sequence alignments are available via anonymous ftp.     

Identification of a structural motif of 23S rRNA interacting with 5S rRNA.

To identify RNA motifs interacting with 5S rRNA, a systematic evolution of ligands by exponential enrichment experiment was applied. Some of the resulting RNA aptamers contained a consensus sequence similar to the sequence in the loop region of helix 89 of 23S rRNA. We show that the synthetic helix 89 RNA motif indeed interacted with 5S rRNA and that the region around loop B of 5S rRNA was involved in this interaction. These results suggest the presence of a novel RNA-RNA interaction between 23S rRNA and 5S rRNA which may play an important role in the ribosome function.     

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).     

Instability of rRNA operons in Bacillus subtilis.

Many laboratory strains of Bacillus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnJ-rrnW or rrnI-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-aroI region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.     

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.     

Unique organization of Leptospira interrogans rRNA genes.

We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA. Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection. Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence. Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L. interrogans. The results revealed the important fact that each rRNA gene is located far from the other rRNA genes. Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.     

Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.     

Variable rRNA gene copies in extreme halobacteria.

Using PFG electrophoresis techniques, we have examined the organization of rRNA gene in halobacterium species. The results show that the organization of rRNA genes among closely related halobacteria is quite heterogeneous. This contrasts with the high degree of conservation of rRNA sequence (1). The possible mechanism of such rRNA gene amplification and its evolutionary implications are discussed.     

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.     

Transposition of a chromosomal segment bounded by redundant rRNA genes into other rRNA genes in Escherichia coli.

We have constructed several mutants of Escherichia coli which have the chromosomal segment between the directly repeated rrnB and rrnE genes deleted from the normal position and transposed into another one of the seven redundant rRNA genes. We have examples where the transposition has been into rrnC, rrnD, rrnG, and rrnH. Included in the evidence for each of these transpositions was the finding that each transposition specifically affected a different one of the seven BamHI-PstI restriction nuclease fragments known to correspond to the seven rrn genes. The transposition mutants were generally healthy, but sensitive mixed-growth experiments revealed that most of them grew somewhat more slowly than the parental control in rich medium. The maximal detrimental effect was a 4 to 5% reduction in growth rate when the transposition of the rrnB-rrnE segment was into rrnG. We have found that a rrnF gene, reported by others to be linked to malA, does not exist in our standard strain, a derivative of Cavalli Hfr. Instead of rrnF, we identified a new rrn gene, rrnH, which mapped near min 5.     

The 5' end of the 18S rRNA can be positioned from within the mature rRNA.

In yeast, the 5' end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 5' region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1. There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease.