Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves.     

The nucleotide sequence of the chloroplast 5S ribosomal RNA from spinach.

Spinacia oleracia cholorplast 5S ribosomal RNA was end-labeled with [32P] and the complete nucleotide sequence was determined. The sequence is: pUAUUCUGGUGUCCUAGGCGUAGAGGAACCACACCAAUCCAUCCCGAACUUGGUGGUUAAACUCUACUGCGGUGACGAU ACUGUAGGGGAGGUCCUGCGGAAAAAUAGCUCGACGCCAGGAUGOH. This sequence can be fitted to the secondary structural model proposed for prokaryotic 5S ribosomal RNAs by Fox and Woese (1). However, the lengths of several single- and double-stranded regions differ from those common to prokaryotes. The spinach chloroplast 5S ribosomal RNA is homologous to the 5S ribosomal RNA of Lemna chloroplasts with the exception that the spinach RNA is longer by one nucleotide at the 3' end and has a purine base substitution at position 119. The sequence of spinach chloroplast 5S RNA is identical to the chloroplast 5S ribosomal RNA gene of tobacco. Thus the structures of the chloroplast 5S ribosomal RNAs from some of the higher plants appear to be almost totally conserved. This does not appear to be the case for the higher plant cytoplasmic 5S ribosomal RNAs.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.     

Ribosomal Ribonucleic Acids of Chloroplastic and Mitochondrial Preparations

RNA prepared from fractions of chloroplasts and mitochondria sedimented at rates characteristic of ribosomal RNA. A predominance of the 18S species was frequently observed in preparations from chloroplasts from romaine lettuce and endive. The usual distribution, a preponderance of the 28S species, was observed in studies on tomato and spinach chloroplasts and mitochondria from mushroom and cauliflower. Comparisons of the base composition of RNA from organelles with their cytoplasmic ribosomal counterparts revealed that the 18S component from romaine lettuce chloroplast was different. A marginally significant difference was observed in the 28S particle from mushroom mitochondria preparations whereas distinct differences, reflected in all the bases, were seen when the 18S component of cauliflower mitochondria preparations was compared with cytoplasmic RNA.     

A novel variety of 4.5 S RNA from Codium fragile chloroplasts

An unusual new chloroplast RNA has been isolated and sequenced in the siphonous green alga, Codium fragile. This RNA is 94 nucleotides in length, has an unusually high A + U content (73%), contains no modified residues, and is as abundant as a single chloroplast tRNA species. Although this RNA is 4.5 S in size, it bears little sequence homology to the widely found and highly conserved 4.5 S RNAs present in the chloroplasts of higher plants. Nevertheless, this RNA may indeed by analogous to the higher plant 4.5 S RNAs, since the Codium 4.5 S RNA has the potential to form a secondary structure which in many respects is remarkably similar to that of known chloroplast 4.5 S RNAs, and hybridization data strongly suggests that the 4.5 S RNA is part of the ribosomal RNA operon, as is the case in higher plant chloroplasts.     

Nucleotide sequence study of mouse 5.8S ribosomal RNA.

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.     

Crystal structure of the ffh and EF-G binding sites in the conserved domain IV of Escherichia coli 4.5S RNA

BACKGROUND: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptide-containing proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation. RESULTS: We have determined by multiple anomalous dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment containing binding sites for both Ffh and EF-G. This fragment consists of three helices connected by a symmetric and an asymmetric internal loop. In contrast to NMR-derived structures reported previously, the symmetric loop is entirely constituted by non-canonical base pairs. These pairs continuously stack and project unusual sets of hydrogen-bond donors and acceptors into the shallow minor groove. The structure can therefore be regarded as two double helical rods hinged by the asymmetric loop that protrudes from one strand. CONCLUSIONS: Based on our crystal structure and results of chemical protection experiments reported previously, we predicted that Ffh binds to the minor groove of the symmetric loop. An identical decanucleotide sequence is found in the EF-G binding sites of both 4.5S RNA and 23S rRNA. The decanucleotide structure in the 4.5S RNA and the ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA might interact with EF-G and function in translating ribosomes.     

Oligonucleotide sequences of pancreatic and T1 ribonuclease digests of 5S ribosomal RNA from mouse cells.

Oligonucleotides produced by complete pancreatic and T1 RNase digestion of 5S ribosomal RNA from a mouse hepatoma, MH 134, have been separated with two-dimensional electrophoresis and their nucleotide sequences determined. Except for the presence of a 5'-terminal diphosphate, these nucleotide sequences were identical with those of KB cells, confirming the identity of the primary structure of 5S RNA between these animals. Both oligonucleotide patterns produced with these enzymes from 5S RNA of the liver were also identical with those of the hepatoma. All these agree with the strong conservation of 5S RNA genes in animal species. However, when 5S ribosomal RNA was extracted from ribosomes which were prepared from microsomal pellet, pancreatic RNase digest contained two trinucleotides (A-G-Cp and G-A-Cp) that were not found in 5S RNA prepared with a one-step procedure. It was concluded that different isolation procedure might indeed cause artifactual fragments on enzymatic digestion due to internal nicks produced during isolation. The significance of 5'-terminal diphosphate in relation to the biosynthesis of 5S ribosomal RNA is also discussed.     

Time of action of 4.5 S RNA in Escherichia coli translation

A new class of suppressor mutants helps to define the role of 4.5 S RNA in translation. The suppressors reduce the requirement for 4.5 S RNA by increasing the intracellular concentration of uncharged tRNA. Suppression probably occurs by prolonging the period in which translating ribosomes have translocated but not yet released the uncharged tRNA, indicating that this is the point at which 4.5 S RNA enters translation. The release of 4.5 S RNA from polysomes is affected by antibiotics that inhibit protein synthesis. The antibiotic-sensitivity of this release indicates that 4.5 S RNA exits the ribosome following translocation and prior to release of protein synthesis elongation factor G. These results indicate that 4.5 S RNA acts immediately after ribosomal translocation. A model is proposed in which 4.5 S RNA stabilizes the post-translocation state by replacing 23 S ribosomal RNA as a binding site for elongation factor G. The 4.5 S RNA-requirement of mutants altered in 23 S ribosomal RNA support this model.     

Accessibility of phosphodiester bonds in the yeast ribosomal 5 S RNA protein complex

The tertiary structure of the protein-associated yeast ribosomal 5 S RNA was examined using ethylnitrosourea reactivity as a probe for phosphodiester bonds. A reduced reactivity was consistently observed in at least nine residues within four distinct regions of the RNA sequence. Seven of these were also observed in three regions of the free RNA molecule while two, A27 and G30, were only present in the ribonucleoprotein complex. The results strongly suggest that the tertiary structure of the free eukaryotic 5 S RNA is largely conserved in the 5 S RNA-protein complex although it appears to be further stabilized in interaction with the ribosomal protein.     

Complete DNA sequence coding for the large ribosomal RNA of yeast mitochondria.

The mitochondrial gene coding for the large ribosomal RNA (21S) has been isolated from a rho- clone of Saccharomyces cerevisiae. A DNA segment of about 5500 base pairs has been sequenced which included the totality of the sequence coding for the mature ribosomal RNA and the intron. The mature RNA sequence corresponds to a length of 3273 nucleotides. Despite the very low guanine-cytosine content (20.5%), many stretches of sequence are homologous to the corresponding Escherichia coli 23S ribosomal RNA. The sequence can be folded into a secondary structure according to the general models for prokaryotic and eukaryotic large ribosomal RNAs. Like the E.coli gene, the mitochondrial gene contains the sequences that look like the eukaryotic 5.8S and the chloroplastic 4.5S ribosomal RNAs. The 5' and 3' end regions show a complementarity over fourteen nucleotides.     

Phylogeny of the 5S ribosomal RNA from Synechococcus lividus II: the cyanobacterial/chloroplast 5S RNAs form a common structural class

The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacterium Synechococcus lividus II has been determined. The sequence is (sequence in text) This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA of S. lividus II has 27 base differences compared with the 5S RNA of the related strain S. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs from S. lividus II, S. lividus III, and spinach chloroplasts are identical, but differ considerably from that of Escherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA of E. coli and these cyanobacterial and chloroplast 5S RNAs.     

Conserved but nonessential interaction of SRP RNA with translation factor EF-G

4.5S RNA is essential for viability of Escherichia coli, and forms a key component of the signal recognition particle (SRP), a ubiquitous ribonucleoprotein complex responsible for cotranslational targeting of secretory proteins. 4.5S RNA also binds independently to elongation factor G (EF-G), a five-domain GTPase that catalyzes the translocation step during protein biosynthesis on the ribosome. Point mutations in EF-G suppress deleterious effects of 4.5S RNA depletion, as do mutations in the EF-G binding site within ribosomal RNA, suggesting that 4.5S RNA might play a critical role in ribosome function in addition to its role in SRP. Here we show that 4.5S RNA and EF-G form a phylogenetically conserved, low-affinity but highly specific complex involving sequence elements required for 4.5S binding to its cognate SRP protein, Ffh. Mutational analysis indicates that the same molecular structure of 4.5S RNA is recognized in each case. Surprisingly, however, the suppressor mutant forms of EF-G bind very weakly or undetectably to 4.5S RNA, implying that cells can survive 4.5S RNA depletion by decreasing the affinity between 4.5S RNA and the translational machinery. These data suggest that SRP function is the essential role of 4.5S RNA in bacteria.     

Phenol Extraction Op 28 S Ribosomal RNA Prom Rat Liver Cytoplasm

A simple and reproducible phenol method for the isolation of 28 S ribosomal RNA from rat liver cytoplasm, free from poly(A)-RNA is described. The procedure is based on the observation that at lower pH of the homogenate (pH 5.5) 28 S ribosomal RNA is extracted, while 18 S ribosomal RNA remains in the interphase layer.

Isolation of pure 28 S or 18 S ribosomal RNA in preparative amounts requires density gradient cen-trifugation or preparative gel electrophoresis. In this communication a rapid and reproducible method for the isolation of 28 S ribosomal RNA is proposed.     

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.     

Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.     

RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.