Structures of complexes of 5S RNA with ribosomal proteins L5, L18 and L25 from Escherichia coli: identification of kethoxal-reactive sites on the 5S RNA.

The topography of Escherichia coli 5S RNA has been examined in the presence of ribosomal proteins L5, L18 and L25 and their different combinations, by comparing the kethoxal modification characteristics of the various RNA-protein complexes with those of the free A-conformer of 5S RNA (Noller &; Garrett, 1979, accompanying paper).Two of the four most reactive guanines, G₁₃ and G₄₁, are unaffected by the protein, in accord with the finding that these are the only two guanines that are accessible in the 50S subunit (Noller &; Herr, 1974). The other two very reactive guanines, G₂₄ and G₆₉, are strongly protected by protein L18, either in the presence or absence of proteins L5 and L25. Protein binding studies with kethoxal-modified 5S RNA provide evidence that one or both of these two guanines are directly involved in the protein-RNA interactions, and this conclusion is supported by the occurrence of guanines in these two positions in all the other sequenced prokaryotic 5S RNAs.The group of less reactive guanines, G₁₆, G₂₃, G₄₄, G₈₆ and G₁₀₇, are protected to some extent by each of the proteins L5, L18 and L25; the strongest effect is with L18. We suggest that this is attributable to a small increase in the conformational homogeneity of the 5S RNA and that L18, in particular, induces some tightening of the RNA structure.Only one guanine, G₆₉, is rendered more accessible by the proteins. This effect is produced by protein L25, which is known to cause some destructuring of the 5S RNA (Bear et al., 1977). There was no other evidence for any destructuring of the 5S RNA. In particular, the sequence 72 to 83, which is complementary to a sequence in 23S RNA (Herr &; Noller, 1975), is not modified. However, in contrast to an earlier report (Erdmann et al., 1973), the conserved sequence G₄₄-A-A-C, which has been implicated in tRNA binding, was not rendered more accessible by the proteins.     

Two distinct conformations of rat liver ribosomal 5S RNA.

Three different conformers of rat liver 5S ribosomal RNA were investigated by partial nuclease cleavage technique using S1 nuclease and cobra venom endoribonuclease (CVE) as conformational probes. Urea-treated and renatured 5S RNA co-migrate on non-denaturing gels, but exhibit distinct differences in their nuclease cleavage patterns. The most prominent differences in S1 nuclease and CVE accessibility of these conformers are located in region 30-50 and around nucleotides 70 and 90. The third form of 5S RNA with higher electrophoretic mobility was generated by EDTA treatment. The cleavage patterns of this 5S RNA conformer are similar to that characteristic for the renatured 5S RNA. The results demonstrate the difference in secondary structure and possibly different tertiary base-pairing interactions of 5S RNA conformers.     

Accessibility of the 5 S RNA in yeast ribosomes

The accessibility of yeast 5 S RNA to modification by diethyl pyrocarbonate was compared in the free 5 S RNA molecule, 60 S subunits and whole ribosomes. All the reactive sites in the free RNA were eliminated or suppressed in ribosomes but two sites. A₅₁ and A₆₄, remained accessible and a slight reactivity was observed at four new sites (G₃₀, G₄₉, G₅₂ and A₇₂). Nucleotide sequences that have been implicated in initiator transfer RNA binding or subunit interactions are not accessible.     

Escherichia coli 5S RNA A and B conformers. Characterisation by enzymatic and chemical methods

The structures of the two stable conformers of Escherichia coli 5 S RNA, the and B form, were compared. Information about the structures were obtained using the methods of limited enzymatic hydrolysis and chemical modification of accessible nucleotides. Base-specific modifications were performed for adenosines and cytidines using diethylpyrocarbonate and dimethylsulfate in combination with a strand-scission reaction at the modified site. Base-specific (RNase T1) as well as conformation-specific (nuclease S1, cobra venom nuclease) enzymes were employed for the limited enzymatic hydrolysis. Clear differences in the accessibility of the two 5 S RNA conformers to the enzymes and the chemical reagents were established and the regions with altered reactivities were localized in the 5 S RNA structure. The results are consistent with the disruption of the secondary structural interactions in helix II and partly in helices III and IV during the transition from the A to the B form. (The numbering of the helices is according to the generally accepted Fox and Woese model.) In addition some regions presumably involved in the tertiary structure are distorted. There is evidence, however, for the new formation of structural regions between two distant sites in the 5 S RNA B form. The results enable us to refine the existing 5 S RNA A-form model and provide insight into the structural dynamics that lead to the formation of the 5 S RNA B form.     

Chiroptical properties of 2,2’‐bioxirane

The two enantiomers of 2,2′‐bioxirane were synthesized, and their chiroptical properties were thoroughly investigated in various solvents by polarimetry, vibrational circular dichroism (VCD), and Raman optical activity (ROA). Density functional theory (DFT) calculations at the B3LYP/aug‐cc‐pVTZ level revealed the presence of three conformers (G₊, G_?, and cis) with Gibbs populations of 51, 44, and 5% for the isolated molecule, respectively. The population ratios of the two main conformers were modified for solvents exhibiting higher dielectric constants (G_? form decreases whereas G₊ form increases). The behavior of the specific optical rotation values with the different solvents was correctly reproduced by time‐dependent DFT calculations using the polarizable continuum model (PCM), except for the benzene for which explicit solvent model should be necessary. Finally, VCD and ROA spectra were perfectly reproduced by the DFT/PCM calculations for the Boltzmann‐averaged G₊ and G_? conformers.     

Relationship between structure and entropy contributions in an anthraquinone mercapto derivative

The structural and thermodynamic properties of an anthraquinone derivative were studied by means of quantum-chemical calculations. Conformational analysis using ab initio and density functional theory methods revealed 14 low-energy conformers. In order to discuss similarities and differences in entropy of the conformers, the rotational and vibrational contributions to entropy were correlated with changes in conformer structure. The component of the moment of inertia perpendicular to the molecular plane gives significant input to ΔS _rot , whereas the largest contributions to the ΔS _vib have vibrations associated with the τ _S1C20 coordinate.

Protection of specific sites in 23 S and 5 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman &; Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are

in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences,

is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G₁₃ is partially protected in 70 S ribosomes. while G₄₁ remains unaffected by subunit association.The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.     

NMR evidence for the existence of two native conformations of 5S RNA

NMR spectra of the non-exchangeable protons in 5S RNA from E. coli show the existence of two distinct conformers of the molecule which meet the operational definition of "A form" or native 5S RNA. Both are easily distinguished spectroscopically from denatured, "B form" 5S RNA. The conditions which interconvert the two A form conformers strongly suggest that the transition between them gives rise to the low temperature optical melting transition first reported in 5S RNA by Kao and Crothers (1).     

Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (T_m) and thermodynamic (−ΔG°) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the −ΔG° and T_m values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson–Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290–350 nm range is correlated linearly with −ΔG° and T_m, but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine–DNA adducts are recognized in nucleotide excision repair.     

Solution conformations of B. subtilis ribosomal 5S RNA: a calorimetric study

The unfolding of B. subtilis 5S RNA is examined by direct calorimetric measurement in the presence of various concentrations of Na⁺ and Mg²⁺. The composite differential scanning calorimetry (DSC) curve is analyzed into 3–5 individual two-state melting transitions. In the absence of added Na⁺ or Mg²⁺, the 5S RNA segments melt together at T_m = 40°C. Addition of Na⁺ stabilizes the molecular structure (T_m = 56°C) and widens the melting temperature range, so that up to five component transitions are observed. Addition of Mg²⁺ alone produces a very stable structure (T_m = 75°C) with highly cooperative melting. Finally, addition of both Na⁺ and Mg²⁺ produces the highest stability (T_m = 76°C). The results are interpreted according to hypothetical secondary and tertiary base-pairing schemes. The conformational changes demonstrated here may facilitate the movement of the protein synthesis machinery during RNA translation.     

Does 5S RNA from E. coli have a pseudoknotted structure?

Chemical modification and limited enzymatic hydrolysis on isolated E. coli 5S RNA have provided informations on the secondary- and tertiary structure compatible with pseudoknotted structures for the A- and B-conformers of the molecule. Changes in the accessibility and reactivity of nucleotides in loop C and at the stem of helix IV in two different 5S RNA conformers are highly suggestive for interactions between bases C35 to C37 with G105 to G107 for the A-form and C38 to U40 and A94 to G96 with additional interactions of C35, C37 with G98 and G100 for the B-form. In both cases the molecules are folded forming pseudoknots and two quasi--continuous double stranded helices with coaxial stacking. The two structures are in perfect agreement with the biochemical data concerning the stability of the molecule and the chemical reactivities of individual nucleotides of the 5S RNA A- and B-conformers.     

Identification and characterization of the packaging proteins of core 40S hnRNP particles

The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C₁ and C₂ of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A₁ and A₂ of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as N^G,N^G-dimethylarginine. The major particle proteins (A₁ and A₂) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A₁, A₂ and B₁) is consistent with a conserved particle structure and function in eucaryotes.     

DFT study of geometrical and vibrational features of a 3′,5′-deoxydisugar-monophosphate (dDSMP) DNA model in the presence of counterions and solvent

The B3LYP/6–31++G* theoretical level was used to study the influence of various hexahydrated monovalent (Li⁺, Na⁺, K⁺) and divalent (Mg²⁺) metal counterions in interaction with the charged PO₂^? group, on the geometrical and vibrational characteristics of the DNA fragments of 3′,5′-dDSMP, represented by four conformers (g⁺g⁺, g⁺t, g^?g^? and g^?t). All complexes were optimized through two solvation models [the explicit model (6H₂O) and the hybrid model (6H₂O/Continuum)]. The results obtained established that, in the hybrid model, counterions (Li⁺, Na⁺, K⁺, Mg²⁺) always remain in the bisector plane of the O1–P–O2 angle. When these counterions are explicitly hydrated, the smallest counterions (Li⁺, Na⁺) deviate from the bisector plane, while the largest counterions (K⁺ and Mg²⁺) always remain in the same plane. On the other hand, the present calculations reveal that the g⁺g⁺ conformer is the most stable in the presence of monovalent counterions, while conformers g⁺t and g^?t are the most stable in the presence of the divalent counterion Mg²⁺. Finally, the hybrid solvation model seems to be in better agreement with the available crystallographic and spectroscopic (Raman) experiments than the explicit model. Indeed, the six conformational torsions of the C4′-C3′-O3′-PO^?₂-O5′-C5′-C4′ segment of all complexes of the g^?g^? conformer in 6H₂O/Continuum remain similar to the available experimental data of A- and B-DNA forms. The calculated wavenumbers of the g⁺g⁺ conformer in the presence of the monovalent counterion and of g^?t conformer in presence of the divalent counterion in the hybrid model are in good agreement with the Raman experimental data of A- and B-DNA forms. In addition, the maximum deviation between the calculated wavenumbers in the 6H₂O/Continuum for the g⁺g⁺ conformer and experimental value measured in an aqueous solution of the DMP-Na⁺ complex, is <1.07% for the PO₂^? (asymmetric and symmetric) stretching modes and <2.03% for the O5′-C5′ and O3′-C3′ stretching modes.

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Graphical abstract dDSMP-(OO)^? Mg²⁺/6W/Continuum