Structure of protein-deficient 50 S ribosomal subunits. Nine core proteins induce the compact conformation of 23 S ribosomal RNA

The complex of 23 S ribosomal RNA with the nine core proteins L2, L3, L4, L13, L17, L20, L21, L22 and L23 obtained either by the disassembly procedure or by reconstitution has been studied by electron microscopy. This complex is found to be very similar to the intact 50 S subunit both in size and in shape.     

Nucleotide sequences of the 5S ribosomal RNA genes and their adjacent regions in Schizosaccharomyces pombe.

The organization of 5S ribosomal RNA (rRNA) genes in the genome of Schizosaccharomyces pombe has been investigated by restriction and hybridization analyses. The 5S rRNA genes were not linked to the other three species of rRNA genes which formed a repeating unit of 6.9 megadaltons, but located in other regions surrounded by heterogeneous sequences. The 5S rRNA gene organization in S. pombe is therefore different from those in other yeasts; Saccharomyces cerevisiae and Torulopsis utilis. Four restriction segments of different sizes each containing a single 5S rRNA gene were cloned on a bacterial plasmid, and the sequences in and around the RNA coding regions were determined. In the RNA coding regions, the sequences in four clones were identical with an exception that one residue has been substituted in one clone. In the flanking regions, the sequences were extremely rich in the AT-content and highly heterogeneous. The sequences were also markedly different from those in the corresponding regions of the other two yeasts. THe presence of T-clusters in the regions immediately after the RNA coding sequences was only notable homology among the four clones and the other two yeasts.     

Secondary structure of total protein and 23S RNA in 50S ribosomal subunits and in the isolated state

Optical and sedimentational studies of isolated 23S RNA, total proteins and some RNP-complexes of the 50S subunits were carried out. It is shown that the secondary structure content of 23S RNA in the ribosome is lower than in the isolated state. Ribosomal proteins stabilize the 23S RNA structure and make it more compact. At the same time they cause some unwinding effect on the secondary structure of the 23S RNA and possibly fix some segments of the 23S RNA in the conformation necessary for its function. In turn, the 23S RNA increased somewhat the level of the total ordered secondary structure in the ribosomal proteins. There was no considerable change of the ratio between the alpha- and beta-structures in the proteins.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

The excision of intervening sequences from Salmonella 23S ribosomal RNA

Novel, approximately 90 bp intervening sequences (IVs) were discovered within the 23S rRNA genes of S. typhimurium and S. arizonae. These non-rRNA sequences are transcribed and then excised during rRNA maturation. The rRNA fragments that result from the excision of the extra sequences are not religated. This results in fragmented 23S rRNAs. The excision of one IVS was shown to be catalyzed in vivo and in vitro by ribonuclease III. These IVSs are highly volatile evolutionarily, sometimes occurring in only some of the multiple rRNA operons of a particular cell. The sporadic nature of the occurrence of fragmented rRNAs among closely related organisms argues that such fragmentation is a derived state, not a primitive one. Possible sources of these IVSs, their parallels with internal transcribed spacers and introns in eukaryotes, and their possible roles in the evolutionary process are discussed.     

Extensions of the known sequences at the 3'' and 5'' ends of 23S ribosomal RNA from Escherichia coli, possible base pairing between these 23S RNA regions and 16S ribosomal RNA.

Extensions of the known sequences at both 3' and 5' ends of 23S ribosomal RNA are presented: The 5' terminal is pG-G-U-U-A-A-G-Cp or pG-G-U... G-U-U-A-A-G-Cp, with a very short sequence between Up and Gp and the 3'terminal is G-A-A-C-C-G-A-(G)-G-C-U-U-A-A-C-C-U-UOH. These two terminal regions exhibit a high degree of complementarity. In addition, extensive complementarities are also found between the 5'terminal sequence of 23S RNA and a sequence contained in section A of the 16S ribosomal RNA, and between the 3'terminal sequence of 23S RNA and sequences in sections O and J in the 16S RNA. The degree of complementarity between the two extremities of 23S RNA, and between these extremities and regions of the 16S RNA, is far greater than would be expected on a random basis suggesting a possible involvement of this base-pairing in the functioning of ribosomes. This possibility is discussed.     

Letters to the editor: Accessibility of 5 S RNA in 50 S ribosomal subunits

Only two sites in 5 S RNA react with Kethoxal in 50 S ribosomal subunits. These two sites, G₁₃ and G₄₁, have previously been found to be accessible in free 5 S RNA. Nucleotide sequences which have been suggested as possible binding sites for the T-ψ-C-G loop of tRNA are not accessible.     

Modification of 50S ribosomal subunits withN-bromosuccinimide

The 50S subunits ofEscherichia coli ribosomes were modified with the tryptophan reagentN-bromosuccinimide, and the sulfhydryl groups, the modification of which is accompanied by stimulation of polypeptide synthesis (López-Rivas, A. et al. (1978) Eur. J. Biochem. 92, 121), were regenerated by incubation with simple thiols. This treatment inactivates poly(U)-dependent polyphenylalanine synthesis, peptidyl transferase and elongation factor G-dependent GTPase. Incubation with proteins from untreated 70S ribosomes produces partial reactivation of polyphenylalanine synthesis and GTPase activity. Modification is accompanied by loss of 4–5 tryptophan residues per subunit.Abbreviation SucNBr N-bromosuccinimide     

The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50s ribosomal subunits

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.     

Nucleotide sequences of 5S ribosomal RNA from four oomycete and chytrid water molds.

The nucleotide sequences of the 5S rRNAs of the oomycete water molds Saprolegnia ferax and Pythium hydnosporum and of the chytrid water molds Blastocladiella simplex and Phlyctochytrium irregulare were determined by chemical and enzymatic partial degradation of 3' and 5' end-labelled molecules, followed by gel sequence analysis. The two oomycete sequences differed in 24 positions and the two chytrid sequences differed in 27 positions. These pairs differed in a mean of 44 positions. The chytrid sequences clearly most resemble the sequence from the zygomycete Phycomyces, while the oomycete sequences appear to be allied with those from protozoa and slime molds.     

Nucleotide sequences of the 4.5 S and 5 S ribosomal RNA genes from tobacco chloroplasts

Summary The DNA sequences of the 4.5 S and 5 S RNA genes from tobacco chloroplasts have been determined. The coding regions for the mature 4.5 S and 5 S RNAs were identified by sequencing these RNAs. The 4.5 S and 5 S RNA genes are composed of 103 and 121 base pairs respectively. These two genes are separated by the 256 base pair spacer. Several unique features in the spacer and in the region downstream from the 5 S coding region are discussed.     

Nucleotide sequences of three poriferan 5 S ribosomal RNAs.

We have determined the nucleotide sequences of 5 S ribosomal RNAs isolated from the sponges Halichondria panicea, Hymeniacidon sanguinea, and Haliclona oculata. The structures can be fitted in a universal five-helix secondary structure model (De Wachter, Chen and Vandenberghe (1982) Biochimie 64, 311-329) applicable to all 5 S RNAs hitherto sequenced. The base pairing scheme proves to be extremely conserved throughout the metazoan kingdom, yet four slightly different variants of the model may be distinguished among the 5 S RNAs from the seven animal phyla investigated until now.     

16S-23S ribosomal RNA spacer regions of Acetobacter europaeus and A. xylinum, tRNA genes and antitermination sequences

Abstract The 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus DSM 6160, A. xylinum NCIB 11664 and A. xyUnion CL27 were amplified by PCR. Specific PCR products were obtained from each strain and their nucleotide sequences determined. The spacer region of A. europaeus comprises 768 nucleotides (nt), that of A. xylinum 778 nt and that of A. xylinum CL27 759 nt. Genes encoding tRNA^Ile and tRNA^Ala were identified. Putative antitermination sequences were found between the tRNA^Ala sequence and the 5'-terminus of the 23S rRNA coding sequence. The boxA element has the nucleotide sequence TGCTCTTTGATA. Based on hybridization data of digested chromosomal DNA with spacer-specific probes, the copy number of the rrn operons on the chromosome of Acetobacter strains is estimated to be four.     

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.