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Results show that the ribosomal protein Bst S4 binds the 5' domain of Bst 16S rRNA with high affinity. Data indicate that the binding is temperature dependent and requires potassium and magnesium.

Research demonstrates that Bacillus stearothermophilus S4 N-terminus, containing 41 amino acid residues, is highly flexible in solution with no apparent secondary structure. Data indicate that two short segments with highly conserved residues exhibit a transient structural order and interact with RNA and ribosomal proteins.

A study was conducted to analyze the extension of comparative sequence-based predictions to specific interactions between proteins and RNA. The C-terminal RNA binding domain of ribosomal protein L11 and its 58 nucleotide binding regions in 23S rRNA were utilized to carry out the analysis. Results indicated that protein-RNA complexes respond in local and global ways to mutations in or near their interaction surfaces.

Research was conducted to examine the hypothesis that a functional ribosomal RNA tertiary structure involves a base triple interaction. A comparative investigation of more than 550 aligned 23S and 23S-like sequences was performed to determine 2 sets of covariations in the 58 nucleotide RNA domain. UV melting and binding assays were used to show the presence of a base triple in the tertiary structure of the L11 binding region of large subunit ribosomal RNA. Results represent the first example of a base triple involved in the formation of a ribosomal RNA tertiary domain.

UV hyperchromicity and calorimetry has been used to study the unfolding of an mRNA pseudoknot inducing ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus. The two-step melt of the pseudoknot, corresponds to its two helical stems. It is thought that the region of intermediate divalent ion binding affinity could be created by the pseudoknot fold.

The C-terminal domain of the ribosomal protein L11 is responsible for RNA recognition while its N-terminal domain affects cooperation with the thiostrepton antibiotic. The target RNA prevents the C-terminal 77 residues from being digested while thiostrepton and RNA protect the whole protein from degeneration during trypsin digestion. Findings indicate that thiostrepton simulates a ribosomal factor that usually reacts with the Ll1 N-terminal domain.

Transient electric birefringence of 10 artificially synthesized double-stranded RNAs reveals that double-stranded RNA has a persistence length of 720 +/- 70 Angstroms. This is approximately 50% greater than the persistence length of double-stranded DNA, which is 450 to 500 Angstroms. The RNAs ranged in length from 92 to 317 base pairs. The increased stiffness of double-stranded RNA could be caused by hydrogen bonding between the 2'-hydroxyl groups.

Gel electrophoretic mobility experiments help examine the bend and twist associated with the bacterial loop E sequence of 5S ribosomal RNA. A phasing experiment with an A2 bulge and loop E within a single duplex RNA reveals the presence of a torsionally stiff permanent bend in loop E. Another phasing experiment involving loop E between two A2 bulges shows that the helical twist in loop E is overwound compared to a duplex RNA. Ribosomal protein L25 precisely identifies loop E but has no influence on loop twist.

A new type of particular ion-RNA binding has been examined in which a ribosomal RNA tertiary structure, identified by protein and antibiotics, is stabilized by NH4+. NH4+ steadies and sharpens the melting transition connected to tertiary reactions better than other alkali metal cations. The assessment in NH4+ reveals that the attraction of the fragment of RNA for the antibiotic thiostrepton or ribosomal protein L11 is 10 times stronger than when measured in Na+.

A pseudoknot is an RNA structural element formed through the standard base pairing of loop region nucleotides having residues outside the loop. Most of the known pseudoknots belong to the H-type family, where hairpin loop nucleotides are paired with single-stranded regions existing outside the hairpin. Nuclear magnetic resonance methods have revealed that the helical regions of H-type pseudoknots were stacked and had minor perturbations at the stem-loop junctions. Pseudoknots play an important role in the stabilization of compact tertiary structures, the formation of unique RNA structures and conformational switches.

A comparative analysis of bacterial mRNA sequences of Bacillus stearothermophilus were carried out to define the features recognized by binding site of an unusual ribosomal protein complex, L10[(L12).sub.4]. The mRNA and ribosome targets were found to use similar RNA features, held in different structural contexts, to recognize L10[(L12).sub.4], while the ribosome is found to ensure the saturation of its L10[(L12).sub.4] binding site by means of an additional protein-protein interaction.

The free energy of the interaction of [Mg.sup.2+] with partially folded I state RNAs is evaluated and the possibility that [Mg.sup.2+] favors compaction of the I state to a set of conformations with a higher average charge density is studied. Results reveal that compaction appears to enhance [Mg.sup.2+]-I state interaction free energies by 10-20%.

A fluorescence method is used to study the contributions of [Mg.sup.2+] ions to the free energy of folding a pseudoknot RNA tertiary structure. The results have shown that the [Mg.sup.2+]-dependent free energy of folding is sensitive to factors that influence the ensemble of RNA conformations present in the partially unfolded state.

The numbers of mutations in the Escherichia coli 58 nt of rRNA domain that alter the stability of the RNA in the L11-RNA complex from -4 to +9 kcal/mol relative to wild type are examined. The estimated stability has corresponded to only a few ribosomes per bacterial cell dissociated from L11 at any time and hence the selective advantage for any further increase in stability might be very small that can be neglected by other competing selective pressures.

Cations play a large role in stabilizing the native state of RNA in vivo. In addition to Mg2+, putrescine2+ is an abundant divalent cation in bacterial cells, but its effect on the folding of RNA tertiary structure has not been widely explored. In this study, we look at how the stabilities of four structured RNAs, each with a different degree of dependence on K+ and Mg2+, are affected by putrescine2+ relative to Mg2+. Through the use of thermal melts, we observe that (i) at a given concentration, putrescine2+ is less effective than Mg2+ at stabilizing RNA, (ii) the stability imparted to RNA by various diamines is a function of charge density (average separation distance between charges) as well as the flexibility of the counterion, and (iii) when Mg2+ is already present in a buffer, further addition of putrescine2+ may either destabilize or stabilize RNA structure, depending on whether the native RNA does or does not chelate Mg2+ ion, respectively. At ion concentrations likely to be found in vivo, the effect of putrescine2+ on the free energy of folding of an RNA tertiary structure is probably quite small compared to that of Mg2+, but the ability of mixed Mg2+/putrescine2+ environments to (in effect) discriminate between different RNA architectures suggests that, in some cells, the evolution of functional RNA structures may have been influenced by the presence of putrescine2+. [ABSTRACT FROM AUTHOR]

To investigate the mechanism by which urea destabilizes RNA structure, urea-induced unfolding of four different RNA secondary and tertiary structures was quantified in terms of an m-value, the rate at which the free energy of unfolding changes with urea molality. From literature data and our osmometric study of a backbone analogue, we derived average interaction potentials (per square angstrom of solvent accessible surface) between urea and three kinds of RNA surfaces: phosphate, ribose, and base. Estimates of the increases in solvent accessible surface areas upon RNA denaturation were based on a simple model of unfolded RNA as a combination of helical and single-strand segments. These estimates, combined with the three interaction potentials and a term to account for interactions of urea with released ions, yield calculated m-values that are in good agreement with experimental values (200 mm monovalent salt). Agreement was obtained only if single-stranded RNAs were modeled in a highly stacked, A-form conformation. The primary driving force for urea-induced denaturation is the strong interaction of urea with the large surface areas of bases that become exposed upon denaturation of either RNA secondary or tertiary structure, though interactions of urea with backbone and released ions may account for up to a third of the m-value. Urea m-values for all four RNAs are salt-dependent, which we attribute to an increased extension (or decreased charge density) of unfolded RNAs with an increased urea concentration. The sensitivity of the urea m-value to base surface exposure makes it a potentially useful probe of the conformations of RNA unfolded states. [ABSTRACT FROM AUTHOR]

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg2+-RNA chelates could be responsible for a significant fraction of the Mg2+-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg2+ chelation is uncoupled from RNA folding and from unfavorable interactions with Mg2+ ions in other environments. In a S8rner rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg2+ and proteinstabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg2+ concentration, and is partially resolved from the accumulation of Mg2+ in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg2+-RNA interaction free energies over the range of accessed Mg2+ concentrations. At 0.1 mM Mg2+ and 60 mM K+, specific site binding contributes ∼-3 kcal/mol of the total Mg2+ interaction free energy of ∼13 kcal/mol from all sources; at higher Mg2+ concentrations the site-binding contribution becomes a smaller proportion of the total (-4.S vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg ∼-RNA interactions. [ABSTRACT FROM AUTHOR]

There are potentially several ways Mg2+ might promote formation of an RNA tertiary structure: by causing a general "collapse" of the unfolded ensemble to more compact conformations, by favoring a reorganization of structure within a domain to a form with specific tertiary contacts, and by enhancing cooperative linkages between different sets of tertiary contacts. To distinguish these different modes of action, we have studied Mg2+ interactions with the adenine riboswitch, in which a set of tertiary interactions that forms around a purine-binding pocket is thermodynamically linked to the tertiary "docking" of two hairpin loops in another part of the molecule. Each of four RNA forms with different extents of tertiary structure were characterized by small-angle X-ray scattering. The free energy of interconversion between different conformations in the absence of Mg2+and the free energy of Mg2+ interaction with each form have been estimated, yielding a complete picture of the folding energy landscape as a function of Mg2+ concentration. At 1 mM Mg2+ (50 mM K+), the overall free energy of stabilization by Mg2+ is large, -9.8 kcal/mol, and about equally divided between its effect on RNA collapse to a partially folded structure and on organization of the binding pocket. A strong cooperative linkage between the two sets of tertiary contacts is intrinsic to the RNA. This quantitation of the effects of Mg2+ on an RNA with two distinct sets of tertiary interactions suggests ways that Mg2+ may work to stabilize larger and more complex RNA structures. [ABSTRACT FROM AUTHOR]

The Mg2+ -induced folding of RNA tertiary structures is readily observed via titrations of RNA with MgCl2. Such titrations are commonly analyzed using a site binding formalism that includes a parameter, the Hill coefficient n, which is sometimes deemed the number of Mg2+ ions bound by the native RNA at specific sites. However, the long-range nature of electrostatic interactions allows ions some distance from the RNA to stabilize an RNA structure. A complete description of all interactions taking place between Mg2+ and an RNA uses a preferential interaction coefficient, Γ2+, which represents the "excess" Mg2+ neutralizing the RNA charge. The difference between Γ2+ for the native and unfolded RNA forms (Δ Γ 2+) is the number of Mg2+ ions "taken up" by an RNA upon folding. Here we determine the conditions under which the Hill coefficient n can be equated to the ion uptake ΔΓ2+ and find that two approximations are necessary: (i) the Mg2+ activity coefficient is independent of concentration during a titration, and (ii) the dependence of ΔΓ2+ on Mg2+ concentration is weak. Titration experiments with a Mg2+-binding dye and an adenine-binding riboswitch were designed to test these approximations. Inclusion of a 30-fold excess of KCl over MgCl2 was sufficient to maintain a constant Mg2+ activity coefficient. We also observed that Mg2+ uptake by the RNA varied from near zero to ~2.6 as the Mg2+ concentration increases over an ~100-fold range. It is possible to determine ΔΓ2+ from Mg2+-RNA titrations, but the values are only applicable to a limited range of solution conditions. [ABSTRACT FROM AUTHOR]

Large ribosomal subunit proteins L10 and L12 form a pentameric protein complex, L10(L12)4, that is intimately involved in the ribosome elongation cycle. Its contacts with rRNA or other ribosomal proteins have been only partially resolved by crystallography. In Escherichia coli, L10 and L12 are encoded from a single operon for which L10(L12)4 is a translational repressor that recognizes a secondary structure in the mRNA leader. In this study, L10(L12)4 was expressed from the moderate thermophile Bacillus stearothermophilus to quantitatively compare strategies for binding of the complex to mRNA and ribosome targets. The minimal mRNA recognition structure is widely distributed among bacteria and has the potential to form a kink—turn structure similar to one identified in the rRNA as part of the L10(L12)4 binding site. Mutations in equivalent positions between the two sequences have similar effects on L10(L12)4—RNA binding affinity and identify the kink—turn motif and a loop AA sequence as important recognition elements. In contrast to the larger rRNA structure, the mRNA apparently positions the kink—turn motif and loop for protein recognition without the benefit of Mg2+-dependent tertiary structure. The mRNA and rRNA fragments bind L10(L12)4 with similar affinity (~108 M-1), but fluorescence binding studies show that a nearby protein in the ribosome, L11, enhances L10(L12)4 binding ~100-fold. Thus, mRNA and ribosome targets use similar RNA features, held in different structural contexts, to recognize L10(L12)4, and the ribosome ensures the saturation of its L10(L12)4 binding site by means of an additional protein-protein interaction. [ABSTRACT FROM AUTHOR]

Investigates the conformation of the symmetric eubacterial loop E sequence of 5S ribosomal RNA using gel electrophoretic mobility measurements. Description of materials and methods of study; Results of investigation; Discussion of results; References.

Cations play a large role in stabilizing the native state of RNA in vivo. In addition to Mg²⁺, putrescine²⁺ is an abundant divalent cation in bacterial cells, but its effect on the folding of RNA tertiary structure has not been widely explored. In this study, we look at how the stabilities of four structured RNAs, each with a different degree of dependence on K⁺ and Mg²⁺, are affected by putrescine²⁺ relative to Mg²⁺. Through the use of thermal melts, we observe that (i) at a given concentration, putrescine²⁺ is less effective than Mg²⁺ at stabilizing RNA, (ii) the stability imparted to RNA by various diamines is a function of charge density (average separation distance between charges) as well as the flexibility of the counterion, and (iii) when Mg²⁺ is already present in a buffer, further addition of putrescine²⁺ may either destabilize or stabilize RNA structure, depending on whether the native RNA does or does not chelate Mg²⁺ ion, respectively. At ion concentrations likely to be found in vivo, the effect of putrescine²⁺ on the free energy of folding of an RNA tertiary structure is probably quite small compared to that of Mg²⁺, but the ability of mixed Mg²⁺/putrescine²⁺ environments to (in effect) discriminate between different RNA architectures suggests that, in some cells, the evolution of functional RNA structures may have been influenced by the presence of putrescine²⁺.

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg(2+)- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ∼-3 kcal/mol of the total Mg(2+) interaction free energy of ∼-13 kcal/mol from all sources; at higher Mg(2+) concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg(2+)-RNA interactions.

There are potentially several ways Mg2+ might promote formation of an RNA tertiary structure: by causing a general "collapse" of the unfolded ensemble to more compact conformations, by favoring a reorganization of structure within a domain to a form with specific tertiary contacts, and by enhancing cooperative linkages between different sets of tertiary contacts. To distinguish these different modes of action, we have studied Mg2+ interactions with the adenine riboswitch, in which a set of tertiary interactions that forms around a purine-binding pocket is thermodynamically linked to the tertiary "docking" of two hairpin loops in another part of the molecule. Each of four RNA forms with different extents of tertiary structure were characterized by small-angle X-ray scattering. The free energy of interconversion between different conformations in the absence of Mg2+ and the free energy of Mg2+ interaction with each form have been estimated, yielding a complete picture of the folding energy landscape as a function of Mg2+ concentration. At 1 mM Mg2+ (50 mM K+), the overall free energy of stabilization by Mg2+ is large, -9.8 kcal/mol, and about equally divided between its effect on RNA collapse to a partially folded structure and on organization of the binding pocket. A strong cooperative linkage between the two sets of tertiary contacts is intrinsic to the RNA. This quantitation of the effects of Mg2+ on an RNA with two distinct sets of tertiary interactions suggests ways that Mg2+ may work to stabilize larger and more complex RNA structures.

The Mg(2+)-induced folding of RNA tertiary structures is readily observed via titrations of RNA with MgCl(2). Such titrations are commonly analyzed using a site binding formalism that includes a parameter, the Hill coefficient n, which is sometimes deemed the number of Mg(2+) ions bound by the native RNA at specific sites. However, the long-range nature of electrostatic interactions allows ions some distance from the RNA to stabilize an RNA structure. A complete description of all interactions taking place between Mg(2+) and an RNA uses a preferential interaction coefficient, Gamma(2+), which represents the "excess" Mg(2+) neutralizing the RNA charge. The difference between Gamma(2+) for the native and unfolded RNA forms (DeltaGamma(2+)) is the number of Mg(2+) ions "taken up" by an RNA upon folding. Here we determine the conditions under which the Hill coefficient n can be equated to the ion uptake DeltaGamma(2+) and find that two approximations are necessary: (i) the Mg(2+) activity coefficient is independent of concentration during a titration, and (ii) the dependence of DeltaGamma(2+) on Mg(2+) concentration is weak. Titration experiments with a Mg(2+)-binding dye and an adenine-binding riboswitch were designed to test these approximations. Inclusion of a 30-fold excess of KCl over MgCl(2) was sufficient to maintain a constant Mg(2+) activity coefficient. We also observed that Mg(2+) uptake by the RNA varied from near zero to approximately 2.6 as the Mg(2+) concentration increases over an approximately 100-fold range. It is possible to determine DeltaGamma(2+) from Mg(2+)-RNA titrations, but the values are only applicable to a limited range of solution conditions.