Structure Of Ribosomal RNA Research Articles

A compilation of large subunit (23S- and 23S-like) ribosomal RNA structures.

Journal Article A compilation of large subunit (23S- and 23S-like) ribosomal RNA structures Get access Robin R. Gutell, Robin R. Gutell * * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Murray N. Schnare, Murray N. Schnare 1Department of Biochemistry, Dalhousie UniversityHalifax, Nova Scotia B3H 4H7, Canada Search for other works by this author on: Oxford Academic PubMed Google Scholar Michael W. Gray Michael W. Gray * 1Department of Biochemistry, Dalhousie UniversityHalifax, Nova Scotia B3H 4H7, Canada * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 20, Issue suppl, 11 May 1992, Pages 2095–2109, https://doi.org/10.1093/nar/20.suppl.2095 Published: 11 May 1992

Characterization of the rDNA unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonas foetus

The ribosomal RNA gene unit of the protozoan parasite Tritrichomonas foetus has been cloned and analyzed. Southern blot analysis of the genomic DNA showed that the ribosomal RNA gene unit is organized as a tandem head to tail repeat with a unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed the presence of 12 copies of the ribosomal RNA gene unit. The lengths of the small subunit ribosomal RNA and 5.8S ribosomal RNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T. foetus small subunit ribosomal RNA sequence is one of the shortest eukaryotic small subunit rRNA sequences, similar in length to those from 2 other amitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequence maintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, while truncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA was measured at 2.5 kb.

Unfolding of the tertiary structure of specific tRNA and ribosomal 5S RNA from plants as studied with hydroxyl radicals

Ribosomal 5S RNA is present in all eubacterial and eukaryotic ribosomes. Despite a large amount of experimental data on the primary and secondary structures of these types of molecules, details of their tertiary structure and their precise function in protein biosynthesis are still not known. Recently we have proposed a new model for the tertiary structure of plant 5S rRNA. In this study we applied the Fe(II)-mediated cleavage reaction to test the model. The data presented here provide experimental evidence that in the 5S rRNA molecule only a few nucleotides are buried in the tertiary structure. Similar experiments performed with methionine initiator tRNA gave results which imply the difference in its structure when compared with the X-ray structure of yeast tRNAPhe.

Erratum to : Primary and Secondary Structures of 5S Ribosomal RNA of Nosema bombycis (Nosematidae, Microsporidia)

Erratum to : Primary and Secondary Structures of 5S Ribosomal RNA of <i>Nosema bombycis</i> (Nosematidae, Microsporidia)

Nucleolin promotes secondary structure in ribosomal RNA

The effect of nucleolin on the secondary structure of RNA was studied using circular dichroism (CD). Nucleolin caused decreases in the main positive bands and shifts to higher wavelengths in the CD spectra of synthetic polynucleotides such as poly(G) and poly(A) indicating helix destabilizing activity. In contrast, nucleolin effected increases in signal and shifts to lower wavelengths of the peaks of CD spectra of ribosomal RNA, suggesting enhancement of secondary structure. Another major nucleolar RNA binding protein, B23, had helix destabilizing activity but did not enhance RNA secondary structure. It is proposed that nucleolin promotes formation of secondary structure in preribosomal RNA during the early stages of ribosome biogenesis.

The computer simulation of RNA folding involving pseudoknot formation

The algorithm and the program for the prediction of RNA secondary structure with pseudoknot formation have been proposed. The algorithm simulates stepwise folding by generating random structures using Monte Carlo method, followed by the selection of helices to final structure on the basis of both their probabilities of occurrence in a random structure and free energy parameters. The program versions have been tested on ribosomal RNA structures and on RNAs with pseudoknots evidenced by experimental data. It is shown that the simulation of folding during RNA synthesis improves the results. The introduction of pseudoknot formation permits to predict the pseudoknotted structures and to improve the prediction of long-range interactions. The computer program is rather fast and allows to predict the structures for long RNAs without using large memory volumes in usual personal computer.

A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences

We describe a new computer program that identifies conserved secondary structures in aligned nucleotide sequences of related single-stranded RNAs. The program employs a series of hash tables to identify and sort common base paired helices that are located in identical positions in more than one sequence. The program gives information on the total number of base paired helices that are conserved between related sequences and provides detailed information about common helices that have a minimum of one or more compensating base changes. The program is useful in the analysis of large biological sequences. We have used it to examine the number and type of complementary segments (potential base paired helices) that can be found in common among related random sequences similar in base composition to 16S rRNA from Escherichia coli. Two types of random sequences were analyzed. One set consisted of sequences that were independent but they had the same mononucleotide composition as the 16S rRNA. The second set contained sequences that were 80% similar to one another. Different results were obtained in the analysis of these two types of random sequences. When 5 sequences that were 80% similar to one another were analyzed, significant numbers of potential helices with two or more independent base changes were observed. When 5 independent sequences were analyzed, no potential helices were found in common. The results of the analyses with random sequences were compared with the number and type of helices found in the phylogenetic model of the secondary structure of 16S ribosomal RNA. Many more helices are conserved among the ribosomal sequences than are found in common among similar random sequences. In addition, conserved helices in the 16S rRNAs are, on the average, longer than the complementary segments that are found in comparable random sequences. The significance of these results and their application in the analysis of long non-ribosomal nucleotide sequences is discussed.

Dynamic structure of bacterial ribosomal 5S RNA: Helices II and III of B. megaterium 5S RNA

A possible switch between two conformations, previously observed in an enzymatically cleaved fragment of E. coli 5S ribosomal RNA (a Gram- negative bacterium) containing helices II and III, has been examined by means of proton nuclear magnetic resonance spectroscopy (10–15 ppm) as a function of [Mg 2+] and temperature for an RNase-T1 digested fragment of Bacillus megaterium 5S rRNA (a Gram- positive bacterium) containing the same helices II and III. The conformational changes induced in the fragment are not accompanied by breakage of some base-pairs and formation of others, but rather consist simply of tightening or loosening of helices with retention of existing base-pairs. Helix III is found to be more flexible than helix II. Finally, the loop conformation is conserved over a wide range of Mg 2+ concentration, suggesting that the loop may serve an important role in the biological function of 5S rRNA in ribosomes.

The primary structure of 5S ribosomal RNAs from Magnolia cobus and Magnolia stellata

The primary structure of 5S ribosomal RNAs from Magnolia cobus and Magnolia stellata

Structure detection through automated covariance search

This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential secondary and tertiary structural interactions, but also provides the covariation evidence that supports the proposed bondings, and any counterevidence that can be detected in the known sequences. A small number of previously unknown higher-order structural features have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. We are systematically studying mitochondrial rRNA, seeking tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. To test hypotheses suggested by an examination of our program's output, our colleagues in biology are sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosomal structure.

Structure of synthetic unmethylated 16S ribosomal RNA as purified RNA and in reconstituted 30S ribosomal subunits.

16S ribosomal RNA was made by in vitro transcription of a cloned gene, and its structure was compared to authentic 16S ribosomal RNA. The comparison was made by subjecting the two types of 16S rRNA to chemical reagents that react specifically with unpaired bases and determining the extent of reaction by reverse transcription and gel electrophoresis of the cDNA. In solution, the rRNAs were indistinguishable in their pattern of reactivity, except for a difference at A1408 and many differences in the region between residues 470 and 562. When the 16S rRNAs were reconstituted into 30S ribosomal subunits, these reactivity differences were absent. When the synthetic 16S rRNA was reconstituted into 30S subunits and then extracted and tested in solution, its pattern of chemical reactivity in the 470-562 region was the same as authentic 16S rRNA, but differences in the 1408-1410 region persisted. This study indicates that the synthetic 16S rRNA has a secondary structure in solution similar to a native secondary structure except in two regions, one apparently incorrectly folded during synthesis and the other in which nucleotides which are normally methylated in authentic 16S rRNA may be responsible for a structural difference.

The structure and evolution of archaebacterial ribosomal RNAs

A cladistic analysis of 553 5S rRNA sequences has revealed a Ur-5S rRNA, the ancestor of all present-day 5S rRNA molecules. Previously stated characteristic differences between the eubacterial and eukaryotic molecules, namely, the length base-pairing schemes of helices D, can be used as a marker for the various archaebacterial branches. One model comprises Thermococcus, Thermoplasma, methanobacteria, and halobacteria; a second comprises the Sulfolobales; and a third is represented only by the single organism Octopus Spring species 1. A relaxed selection pressure on helix E with subsequent deletions is observed in Methanobacteriales, Methanococcales, and eubacteria. The secondary structures are supported by enzymatic digestion and chemical modification studies of the 5S rRNAs. Reconstitution of eubacterial 50S ribosomal subunits with 5S rRNA from Halobacterium and Thermoplasma has revealed 100% incorporation, while eukaryotic 5S rRNAs yielded a 50% incorporation. Relevant positions of the small-subunit rRNA are selected to answer the question of the monophyly of archaebacteria. Eight positions account for monophyly, eight for an ancestry of eubacteria with halophile methanogens and eukaryotes with eocytes (paraphyly of archaebacteria), and two for an ancestry of eubacteria with eocytes. A refinement of the neighborliness method of S. Sattath and A. Tversky resulted in a monophyly of archaebacteria when all positions are treated equally and in a paraphyly when tranversions are weighted twice over transitions.

Primary and secondary structure of the 18S ribosomal RNA of the bird spider Eurypelma californica and evolutionary relationships among eukaryotic phyla.

The primary structure of the 18S rRNA of the bird spider Eurypelma californica has been determined in the framework of a study of metazoan phylogeny on the basis of ribosomal RNA structure. A secondary-structure model was derived by comparison of the sequence with that of 43 other eukaryotic small-ribosomal-subunit RNA sequences presently available. This comparison allows a rather detailed secondary-structure pattern to be postulated for a eukaryote-specific area of highly variable sequence and length for which no consensus model has hitherto been attained. A dendrogram, reflecting evolutionary relationships among the 40 eukaryotic species of known 18S rRNA structure, was constructed by a matrix method selecting the best-fitting tree on the basis of a least-squares criterion. The tree shows an early divergence of a microsporidium, an euglenoid, kinetoplastids and a slime mold. Among the remaining species, two main clusters are distinguishable, one comprising the Ciliata, the other comprising Metazoa, green plants, fungi and several protists. Among the Metazoa, the three phyla presently investigated, viz. Chordata, Arthropoda and Nemathelminthes, are distinguishable as three separate lines of descent.

Evolution of the secondary structures and compensatory mutations of the ribosomal RNAs of Drosophila melanogaster.

This paper examines the effects of DNA sequence evolution on RNA secondary structures and compensatory mutations. Models of the secondary structures of Drosophila melanogaster 18S ribosomal RNA (rRNA) and of the complex between 2S, 5.8S, and 28S rRNAs have been drawn on the basis of comparative and energetic criteria. The overall AU richness of the D. melanogaster rRNAs allows the resolution of some ambiguities in the structures of both large rRNAs. Comparison of the sequence of expansion segment V2 in D. melanogaster 18S rRNA with the same region in three other Drosophila species and the tsetse fly (Glossina morsitans morsitans) allows us to distinguish between two models for the secondary structure of this region. The secondary structures of the expansion segments of D. melanogaster 28S rRNA conform to a general pattern for all eukaryotes, despite having highly divergent sequences between D. melanogaster and vertebrates. The 70 novel compensatory mutations identified in the 28S rRNA show a strong (70%) bias toward A-U base pairs, suggesting that a process of biased mutation and/or biased fixation of A and T point mutations or AT-rich slippage-generated motifs has occurred during the evolution of D. melanogaster rDNA. This process has not occurred throughout the D. melanogaster genome. The processes by which compensatory pairs of mutations are generated and spread are discussed, and a model is suggested by which a second mutation is more likely to occur in a unit with a first mutation as such a unit begins to spread through the family and concomitantly through the population. Alternatively, mechanisms of proofreading in stem-loop structures at the DNA level, or between RNA and DNA, might be involved. The apparent tolerance of noncompensatory mutations in some stems which are otherwise strongly supported by comparative criteria within D. melanogaster 28S rRNA must be borne in mind when compensatory mutations are used as a criterion in secondary-structure modeling. Noncompensatory mutation may extend to the production of unstable structures where a stem is stabilized by RNA-protein or additional RNA-RNA interactions in the mature ribosome. Of motifs suggested to be involved in rRNA processing, one (CGAAAG) is strongly overrepresented in the 28S rRNA sequence. The data are discussed both in the context of the forces involved with the evolution of multigene families and in the context of molecular coevolution in the rDNA family in particular.

Higher order structure of chloroplastic 5S ribosomal RNA from spinach.

The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.

The emerging three-dimensional structure and function of 16S ribosomal RNA.

The emerging three-dimensional structure and function of 16S ribosomal RNA.

Chemical probing of adenine residues within the secondary structure of rabbit 18S ribosomal RNA.

The location of unpaired adenine residues within the secondary structure of rabbit 18S ribosomal RNA was determined by chemical probing. Naked 18S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked 18S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides [Peattie, D. A., & Gilbert, W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4679-4682]. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of 32P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in 18S rRNA determined by comparative sequence analysis [Chan, Y.-L., Gutell, R., Noller, H. F., & Wool, I. G. (1984) J. Biol. Chem. 259, 224-230]. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit 18S rRNA than for E. coli 16S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian 18S rRNA, as compared with prokaryotic 16S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

Revised primary structure of rabbit 18S ribosomal RNA.

Revised primary structure of rabbit 18S ribosomal RNA.

Probing ribosome structure using short oligodeoxyribonucleotides: the question of resolution

The structure of ribosomal RNA in situ can be probed using short, complementary DNA oligomers. As these oligomers bind to exposed, single-stranded regions of rRNA, the stability of the hybridized complex can be assayed. Differences in binding stability between cDNA probes of similar length and composition may be indicative of the presence of competing structure, such as base-paired rRNA regions, tRNA interactions or protein interactions. In this study the degree to which such interactions can be distinguished is studied. It is found that by using suitable controls, interactions between rRNA and tRNA or rRNA can be discriminated to a resolution of one or two bases. This resolution promises to be important in delineating the higher-order structure of the rRNA.

Structure of 5S rRNA in actinomycetes and relatives and evolution of eubacteria

The primary structure of 5S ribosomal RNA has been determined in five species belonging to the genus Mycobacterium and in Micrococcus luteus. The sequences of 5S RNAs from Actinomycetes and relatives point to the existence in this taxon of a bulge on the helix that joins the termini of the molecule. An attempt was made to reconstruct bacterial evolution from a sequence dissimilarity matrix based on 142 eubacterial 5S RNA sequences and corrected for multiple mutation. The algorithm is based on weighted pairwise clustering, and incorporates a correction for divergent mutation rates, as derived by comparison of sequence dissimilarities with an external reference group of eukaryotic 5S RNAs. The resulting tree is compared with the eubacterial phylogeny built on 16S rRNA catalog comparison. The bacteria for which the 5S RNA sequence is known form a number of clusters also discernible in the 16S rRNA phylogeny. However, the branching pattern leading to these clusters shows some notable discrepancies with the aforementioned phylogeny.