Comparison of eubacterial and eukaryotic 5S RNA structures: a Raman spectroscopic study

Raman spectra of eubacterial ribosomal 5S RNAs of Escherichia coli, Bacillus subtilis and Thermus thermophilis and of eukaryotic 5S RNAs of yeast and rat liver have been compared. The spectra show a very high and comparable regularity in the ribophosphate backbone as indicated by the ratio 1.67±0.03 for I₈₁₂/I₁₁₀₀ in all samples. The 5S RNAs studied have a similar degree of stacking of the G, A and pyrimidine bases. A high percentage of base-paired U residues between 43 and 66% is indicated. Conformational alterations occurring in 5S RNAs in the presence of Mg²⁺ ions between 20 and 50^*C are localized mainly in the region of loop II of the molecule. The implications of these results for the 5S RNA structure are discussed.     

Nucleotide sequence and structure of cytoplasmic 5S RNA and 5.8S RNA of Chlamydomonas reinhardii.

Three small RNAs of the cytoplasmic 8OS ribosomes of the green unicellular alga Chlamydomonas reinhardii have been sequenced. They include two species of ribosomal 5S RNA, a major and a minor one of 122 and 121 nucleotides respectively, which differ from each other by 17 bases, and also the ribosomal 5.8S RNA of 156 nucleotides. Novel structural features can be recognized in the 5S RNAs of C. reinhardii by a comparison with published 5S RNA sequences. In addition the secondary structure of these small RNA molecules has been examined using a newly developed method based on differential nuclease susceptibility.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

Evolutionary changes in the higher order structure of the ribosomal 5S RNA.

Comparative studies have been undertaken on the higher order structure of ribosomal 5S RNAs from diverse origins. Competitive reassociation studies show that 5S RNA from either a eukaryote or archaebacterium will form a stable ribonucleoprotein complex with the yeast ribosomal 5S RNA binding protein (YL3); in contrast, eubacterial RNAs will not compete in a similar fashion. Partial S1 ribonuclease digestion and ethylnitrosourea reactivity were used to probe the structural differences suggested by the reconstitution experiments. The results indicate a more compact higher order structure in eukaryotic 5S RNAs as compared to eubacteria and suggest that the archaebacterial 5S RNA contains features which are common to either group. The potential significance of these results with respect to a generalized model for the tertiary structure of the ribosomal 5S RNA and to the heterogeneity in the protein components of 5S RNA-protein complexes are discussed.     

An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition.

RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 10(12) random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA) of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 microM by equilibrium filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil. The K d values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding.     

RNA associated with nonhistone chromosomal proteins of dog liver

The nonhistone chromosomal proteins were separated on Sephadex G-200 into 3 fractions of which two were associated with 3S RNA. The RNA eluted with fraction I (guanine + cytosine content 54%) is tightly bound to the proteins from which it can be separated only after digestion with pronase. The RNA associated with fraction III (guanine + cytosine content 64%) can be separated from the proteins directly by chromatography on DEAE-Sephadex A 25. No dihydropyrimidines have been detected in any of the two RNAs.     

Idealized atomic coordinates of yeast phenylalanine transfer RNA.

The atomic coordinates are given for yeast phenylalanine transfer RNA in the orthorhombic crystal form. The structure has been refined by fitting to successively improved electron density maps at 2.7 Å resolution. The model fitting has been accomplished by using an interactive computer graphics system to minimize the errors inherent in manual model building and coordinate measurements, using an optical comparator. The atomic coordinates have then been “idealized” to make bond distances, bond angles, steric conformation and non-bonded contacts close to standard values, while constraining the model to fit the electron density maps.     

Changes in the conformation and stability of 5 S RNA upon the binding of ribosomal proteins.

The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylation patterns of mycoplasma transfer and ribosomal ribonucleic acid.

The methylation patterns of transfer and ribosomal ribonucleic acid (RNA) from two mycoplasmas, Mycoplasma capricolum and Acholeplasma laidlawii, have been examined. The transfer RNA from the two mycoplasmas resembled that of other procaryotes in degree of methylation and general diversity of methylated nucleotides, and bore particular resemblance to Bacillus subtilis transfer RNA. The only unusual feature was the absence of m5U from M. capricolum transfer RNA. The methylation patterns of the mycoplasma 16S RNAs were also typically procaryotic, retaining the methylated residues previously shown to be highly conserved among eubacterial 16S RNAs. The mycoplasma 23S RNA methylation patterns were, on the other hand, quite unusual. M. capricolum 23S RNA contained only four methylated residues in stoichiometric amounts, all of which were ribose methylated. A. laidlawii 23S RNA contained the same ribose-methylated residues, plus in addition approximately six m5U residues. These findings are discussed in relation to the phylogenetic status of mycoplasma, as well as the possible role of RNA methylation.     

Accessible and inaccessible bases in yeast phenylalanine transfer RNA as studied by chemical modification.

The selective modification of cytidine, uridine, guanosine and dihydrouridine residues in ³²P-labelled yeast phenylalanine transfer RNA has been studied by the use of specific reagents.The selective modification of cytidine residues with the reagent methoxyamine is described. Of the six cytidines in the single-stranded regions of the cloverleaf formula, only two are completely reactive, C74 and C75 at the 3′-terminus. Cm32 in the anticodon loop is reactive to only a small extent.The selective modifications of uridine and guanosine residues with 1-cyclohexyl 3-[2-morpholino(4)-ethyl] carbodiimide methotosylate, is described. The reagent is also shown to be reactive with dihydrouridine. In the single-stranded regions of the secondary structure of yeast phenylalanine transfer RNA there are 16 base residues which this reagent could be specific for. However, only G20, Gm34 and U47 are extensively modified, whilst U33 and D16 are partially modified. G18 is modified to a very small extent.The results obtained in this study are also in good agreement with previous chemical modification studied by other workers, carried out on unlabelled yeast phenylalanine transfer RNA using different reagents to the ones described here.The pattern of chemical modification is compared with the three-dimensional structure obtained by an X-ray crystallographic analysis of the same tRNA species. The correlation between exposed regions of the model and the regions of chemical reactivity are everywhere consistent.     

Cross section calculations for electron scattering from DNA and RNA bases

Differential and integral cross sections for elastic electron collisions with uracil, cytosine, guanine, adenine and thymine have been calculated using the independent atom method with a static-polarization model potential for incident energies ranging from 50 to 4000 eV. Total cross sections for single electron-impact ionization of selected DNA and RNA bases have also been calculated with the binary-encounter-Bethe model from the ionization threshold up to 5000 eV. Cross sections within the investigated energy range, can be related to the molecular symmetry, the number of target electrons and molecular size; elastic and ionization processes are most efficient for guanine and adenine molecules, while the lowest cross sections were obtained for the uracil molecule. The ionization cross sections for cytosine, thymine, adenine and guanine are compared with those recently obtained with a semi-classical and binary-encounter-Bethe formalisms. No theoretical and experimental data for elastic electron scattering from DNA and RNA bases are available, but comparisons with calculations for molecules of similar size and geometry allows the validity of the theoretical approach to be verified.     

Correlation between chemical modification and surface accessibility in yeast phenylalanine transfer RNA

Chemical reactivities of the functional groups of yeast phenylalanine transfer RNA are compared with surface accessibilities of the groups calculated with various probe radii representing effective radii of the chemical reagents used. We observe 97% agreement with the hypothesis that the chemically modified bases are those with the greatest surface accessibility. This overall strong correlation supports the conclusion that base exposure in an important determinant of chemical modification in this polynucleotide.     

Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs.

The RNA genome of poliovirus hybridizes to 28S and 18S rRNAs of higher eukaryotes under stringent conditions. The hybridization detected by Northern blot analyses is specific since little or no signal was detected for yeast or prokaryotic rRNAs or other major cellular RNAs. Southern blot analysis of DNA clones of mouse rRNA genes leads us to conclude that several regions of 28S rRNA, and at least one region in 18S rRNA, are involved in the hybridization to polio RNA, and that G/C regions are not responsible for this phenomenon. We have precisely mapped one of these hybridizing regions in both molecules. Computer analysis confirms that extensive intermolecular base-pairing (81 out of 104 contiguous bases in the rRNA strand) could be responsible for this one particular site of interaction (polio genome, bases 5075-5250; 28S rRNA, bases 1097-1200). We discuss the possible functional and/or evolutionary significance of this novel type of interaction.     

Sequence and conformation of 5 S RNA from Chlorella cytoplasmic ribosomes: comparison with other 5 S RNA molecules

The sequence of Chlorella cytoplasmic 5 S RNA has been determined by fingerprinting techniques. Partial digests were fractionated by a two-dimensional acrylamide gel electrophoretic technique, which indicates whether specific fragments are paired in the molecule. In this way, the four main base-paired regions of the molecule were located. The sequence of Chlorella cytoplasmic 5 S RNA is related to, but different from, that of other eukaryotic 5 S RNAs: it shows approximately 60% homology with vertebrate 5 S RNA and 40% homology with yeast 5 S RNA. In some respects the conformation of the molecule in solution is quite different from that of other sequenced 5 S RNAs: in particular, the highly accessible region found around position 40 in all other 5 S RNAs (prokaryotic and eukaryotic) does not exist in this molecule.     

Potential of mean force calculations of the stacking-unstacking process in single-stranded deoxyribodinucleoside monophosphates.

The free energy of the stacking-unstacking process of deoxyribodinucleoside monophosphates in aqueous solution has been investigated by potential of mean force calculations along a reaction coordinate, defined by the distance between the glycosidic nitrogen atoms of the bases. The stacking-unstacking process of a ribodinucleoside monophosphate was observed to be well characterized by this coordinate, which has the advantage that it allows for a dynamical backbone and flexible bases. All 16 naturally occurring DNA dimers composed of the adenine, cytosine, guanine, or thymine bases in both the 5' and the 3' positions were studied. From the free-energy profiles we observed the deepest minima for the stacked states of the purine-purine dimers, but good stacking was also observed for the purine-pyrimidine and pyrimidine-purine dimers. Substantial stacking ability was found for the dimers composed of a thymine base and a purine base and also for the deoxythymidylyl-3',5'-deoxythymidine dimer. Very poor stacking was observed for the dCpdC dimer. Conformational properties and solvent accessibility are discussed for the stacked and unstacked dimers. The potential of mean force profiles of the stacking-unstacking process for the DNA dimers are compared with the RNA dimers.     

Polyribosome Profiles and Polyribosome-Associated RNA of Friend Leukaemia Cells Following DMSO-induced Differentiation

Differentiation along erythroid lines is greatly increased when Friend leukaemia cells (FLC) are grown in the presence of dimethylsulfoxide (DMSO). Maturation is accompanied by alterations in the polyribosome profiles of the DMSO-treated cells as compared to that of control cells grown for the same periods of time. The ribosomes of the differentiating cells are present primarily as trimers, tetramers and pentamers, with a low proportion of larger polysomes. Polyribosomes consisting of more than five ribosomes are much more common in the control untreated FLC.
Agarose-acrylamide gel electrophoresis of radioactive uridine-labelled polysome-associated RNA from control and DMSO-treated cells demonstrates 4, 5, 5.5, 7, 9, 18 and 28S RNA species, as well as a series of RNAs which range from approximately 10S to 18S. The 5, 5.5, 18 and 28S RNAs have been tentatively identified as ribosomal RNAs, and the 4S and 7S RNAs may be transfer RNA and viral RNA respectively. The 9S RNA is probably the mRNA for histone, since its synthesis is inhibited in cells incubated with hydroxyurea. RNA which co-electrophoreses with the presumptive globin mRNA has been identified both in control, and in DMSO-treated cells. Although alterations in polyribosome patterns of DMSO-treated cells, as compared to control cells, have been readily demonstrable, no differences in the polyribosome-associated RNAs have been detected so far.     

Isolation and characterization of two linked mouse U1b small nuclear RNA genes.

A 6.9 kilobase Eco R1 fragment containing genes for two U1 RNAs has been isolated from a library of mouse DNA. The two genes code for an RNA which is very similar, if not identical, to mouse U1b RNA as judged by S1 nuclease mapping. This RNA is one base longer than the mouse U1a RNA, human U1 RNA, and rat U1 RNA and differs in six nucleotide substitutions from rat U1 RNA. The two genes are five kilobases apart and the U1 RNAs are coded for on opposite strands of the DNA with the 5' ends juxtaposed. The sequences flanking the genes are identical for 700 bases 5' to the gene and at least 80 bases 3' to the gene.