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PubMed1996

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.

Amino Acid Sequence↗

PubMed1996

The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3' untranslated region.

Controls that act at both transcriptional and posttranscriptional levels assure that globin genes are highly expressed in developing erythroid cells. The extraordinary stabilities of alpha- and beta-globin mRNAs permit globin proteins to accumulate to substantial levels in these cells, even in the face of physiologic transcriptional silencing. Structural features that determine alpha-globin mRNA stability have recently been identified within its 3'UTR; in contrast, the structural features that determine beta-globin mRNA stability remain obscure. The current study begins to define the structural basis for beta-globin mRNA stability. Two tandem antitermination mutations are introduced into the wild-type human beta-globin gene that permit ribosomes to read into the 3'UTR of the encoded beta-globin mRNA. The readthrough beta-globin mRNA is destabilized in cultured erythroid cells, indicating that, as in human alpha-globin mRNA, an unperturbed 3'UTR is crucial to maintaining mRNA stability. Additional experiments show that the beta-globin and alpha-globin mRNA 3'UTRs provide equivalent levels of stability to a linked beta-globin mRNA coding region, suggesting a parallel in their functions. However, destabilization of the antiterminated beta-globin mRNA is independent of active translation into the 3'UTR, whereas translation into the alpha-globin mRNA 3'UTR destabilizes a linked beta-globin coding region in a translationally dependent manner. This indicates that the alpha- and beta-globin 3'UTRs may stabilize linked mRNAs through distinct mechanisms. Finally, it is shown that neither of the two mutations that, in combination, destabilize the beta-globin mRNA have any effect on beta-globin mRNA stability when present singly, suggesting potential redundancy of stabilizing elements. In sum, the current study shows that a functionally intact beta-globin mRNA 3'UTR is crucial to maintaining beta-globin mRNA stability and provides a level of stability that is functionally equivalent to, although potentially mechanistically distinct from, the previously characterized alpha-globin mRNA 3'UTR stability element.

Animals↗

PubMed1990

Secondary structure at the 3' terminal region of RNA coliphages: comparison with tRNA.

Secondary structure models for the 3' non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes. We report the following observations. (1) In contrast to the coding regions, the structure at the 3' terminus is characterized by stable regular helices. We note the occurrence of the loop sequences 5'-GUUCGC and 5'-CGAAAG, that are reported to confer exceptional stability to stem structures. These features are probably present to promote the segregation of mother and daughter strands during replication. (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability. (3) We have compared the structure of phage RNA with tRNA. Overall similarity is low, but one common element may exist. It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the T psi C arm and the aminoacyl acceptor arm in tRNA. As in tRNA, this structure element starts after the fourth nucleotide from the 3' end. (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix. We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA. The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein. This common binding site may serve as a switch from replication to packaging.

Anticodon↗

PubMed2004

Secondary structure as a functional feature in the downstream region of mammalian polyadenylation signals.

Secondary structure within the downstream region of mammalian polyadenylation signals has been proposed to perform important functions. The simian virus 40 late polyadenylation signal (SVLPA) forms alternate secondary structures in equilibrium. Their formation correlates with cleavage-polyadenylation efficiency (H. Hans and J. C. Alwine, Mol. Cell. Biol. 20:2926-2932, 2000; M. I. Zarudnaya, I. M. Kolomiets, A. L. Potyahaylo, and D. M. Hovorun, Nucleic Acids Res. 3:1375-1386, 2003), and oligonucleotides that disrupt the secondary structure inhibit in vitro cleavage. To define the important features of downstream secondary structure, we first minimized the SVLPA by deletion, forming a downstream region with fewer, and more stable, stem-loop structures. Specific mutagenesis showed that both stem stability and loop size are important functional features of the downstream region. Stabilization of the stem, thus minimizing alternative structures, decreased cleavage efficiency both in vitro and in vivo. This was most deleterious when the stem was stabilized at the base of the loop, constraining loop size by inhibiting breathing of the stem. The significance of loop size was supported by mutants that showed increased cleavage efficiency with increased loop size and vice versa. A loop of at least 12 nucleotides promoted cleavage; U richness in the loop also promoted cleavage and was particularly important when the stem was stabilized. A mutation designed to eliminate downstream secondary structure still formed many relatively weak alternative structures in equilibrium and retained function. The data suggest that although the downstream region is very important, its structure is quite malleable and is able to tolerate significant mutation within a wide range of primary and secondary structural features. We propose that this malleability is due to the enhanced ability of GU- and U-rich downstream elements to easily form secondary structures with surrounding sequences.

Animals↗

PubMed2000

A theoretical model for the prediction of sequence-dependent nucleosome thermodynamic stability.

A theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence is advanced. The model is based on a statistical mechanical approach, which allows the calculation of the canonical ensemble free energy involved in the competitive nucleosome reconstitution. It is based on the hypothesis that nucleosome stability mainly depends on the bending and twisting elastic energy to transform the DNA intrinsic superstructure into the nucleosomal structure. The ensemble average free energy is calculated starting from the intrinsic curvature, obtained by integrating the dinucleotide step deviations from the canonical B-DNA and expressed in terms of a Fourier series, in the framework of first-order elasticity. The sequence-dependent DNA flexibility is evaluated from the differential double helix thermodynamic stability. A large number of free-energy experimental data, obtained in different laboratories by competitive nucleosome reconstitution assays, are successfully compared to the theoretical results. They support the hypothesis that the stacking energies are the major factor in DNA rigidity and could be a measure of DNA stiffness. A dual role of DNA intrinsic curvature and flexibility emerges in the determination of nucleosome stability. The difference between the experimental and theoretical (elastic) nucleosome-reconstitution free energy for the whole pool of investigated DNAs suggests a significant role for the curvature-dependent DNA hydration and counterion interactions, which appear to destabilize nucleosomes in highly curved DNAs. This model represents an attempt to clarify the main features of the nucleosome thermodynamic stability in terms of physical-chemical parameters and suggests that in molecular systems with a large degree of complexity, the average molecular properties dominate over the local features, as in a statistical ensemble.

Base Sequence↗

PubMed2004

A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility.

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair(NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.

Animals↗

PubMed1979

A relationship between DNA helix stability and recognition sites for RNA polymerase.

The RNA polymerase binding sites on the DNA of (i) the aroE-trkA-spc segment of the Escherichia coli genome, (ii) transposon Tn3, (iii) plasmid ColE1, and (iv) coliphage lambda were mapped by electron microscopy, with the use of the BAC technique; these maps were compared with the maps of the early-melting regions for the same genomes. The results indicate that in all these cases the binding sites for the E. coli RNA polymerase lie preferentially in the early melting regions of DNA. These data indicate that helix stability may be an important feature of the multipartite nature of the promoter structure.

DNA, Bacterial↗

PubMed2005

A stability pattern of protein hydrophobic mutations that reflects evolutionary structural optimization.

We have determined the effect of mutations involving isoleucine and valine (i.e., mutations I-->V and V-->I) on the stability of Escherichia coli thioredoxin. Despite the similarity in chemical structure (V and I differ only in a methyl group), we find that many environments are optimized to a significant extent for either V or I. We find, furthermore, that a plot of effect of hydrophobic mutations on stability versus packing density shows a strikingly simple pattern that clearly reflects evolutionary structural optimization. The existence of such patterns suggests the possibility of rationalizing (and perhaps even predicting) mutation effects on protein stability on the basis of evolutionary models. By "evolutionary model" we specifically refer in this context to a model for mutation effects on stability in which certain physical features of the mutated residue environments are evaluated from an assumption regarding how such environments have been selected during protein evolution (as opposed to a purely "physical model" in which those features would be derived from some kind of energetics analysis of the protein structural characteristics). To illustrate this novel approach and provide general guidelines for its application, we develop here a simple evolutionary model that successfully explains the effect of the I<-->V mutations on thioredoxin stability.

Algorithms↗

PubMed2000

Cold adapted enzymes.

The number of reports on enzymes from cold adapted organisms has increased significantly over the past years, and reveals that adaptive strategies for functioning at low temperature varies among enzymes. However, the high catalytic efficiency at low temperature seems, for the majority of cold active enzymes, to be accompanied by a reduced thermal stability. Increased molecular flexibility to compensate for the low working temperature, is therefore still the most dominating theory for cold adaptation, although there also seem to be other adaptive strategies. The number of experimentally determined 3D structures of enzymes possessing cold adaptation features is still limited, and restricts a structural rationalization for cold activity. The present summary of structural characteristics, based on comparative studies on crystal structures (7), homology models (7), and amino acid sequences (24), reveals that there are no common structural feature that can account for the low stability, increased catalytic efficiency, and proposed molecular flexibility. Analysis of structural features that are thought to be important for stability (e.g. intra-molecular hydrogen bonds and ion-pairs, proline-, methionine-, glycine-, or arginine content, surface hydrophilicity, helix stability, core packing), indicates that each cold adapted enzyme or enzyme system use different small selections of structural adjustments for gaining increased molecular flexibility that in turn give rise to increased catalytic efficiency and reduced stability. Nevertheless, there seem to be a clear correlation between cold adaptation and reduced number of interactions between structural domains or subunits. Cold active enzymes also seem, to a large extent, to increase their catalytic activity by optimizing the electrostatics at and around the active site.

Adaptation, Biological↗

PubMedDate not supplied

The best of all worlds or the best possible world? Developmental constraint in the evolution of beta-tubulin and the sperm tail axoneme.

Through evolutionary history, some features of the phenotype show little variation. Stabilizing selection could produce this result, but the possibility also exists that a feature is conserved because it is developmentally constrained--only one or a few developmental mechanisms can produce that feature. We present experimental data documenting developmental constraint in the assembly of the motile sperm tail axoneme. The 9+2 microtubule architecture of the eukaryotic axoneme has been deeply conserved. We argue that the quality of motility supported by axonemes with this morphology explains their long conservation, rather than a developmental necessity for the 9+2 architecture. However, our functional tests in Drosophila spermatogenesis reveal considerable constraint in the coevolution of testis-specific beta-tubulin and the sperm tail axoneme. The evolution of testis beta-tubulins used in insect sperm tail axonemes is highly punctuated, indicating some pressure acting on their evolution. We provide a mechanistic explanation for their punctuated evolution by testing structure-function relationships between testis beta-tubulin and the motile axoneme in D. melanogaster. We discovered that a highly conserved sequence feature of beta-tubulins used in motile axonemes is needed to specify central pair formation. Second, our data suggest that cooperativity in the function of internal beta-tubulin amino acids is needed to support the long axonemes characteristic of Drosophila sperm tails. Thus, central pair formation constrains the evolution of the axoneme motif, and intramolecular cooperativity makes the evolution of the internal residues path dependent, which slows their evolution. Our results explain why a highly specialized beta-tubulin is needed to construct the Drosophila sperm tail axoneme. We conclude that these constraints have fixed testis-specific beta-tubulin identity in Drosophila.

Animals↗

PubMed1995

Structural and functional effects of multiple mutations at distal sites in cytochrome c.

Multiple mutations at distally located sites have been introduced into yeast iso-1 cytochrome c to determine the contributions of three amino acids to the structural and functional properties of this protein. The mutant proteins, for which high-resolution structures were determined, included all possible combinations of the substitutions Arg38Ala, Asn52Ile, and Phe82Ser. Arg38, Asn52, and Phe82 are all conserved in the primary sequences of eukaryotic cytochromes c and have been shown to significantly affect several properties of these proteins including protein stability, heme reduction potential, and oxidation state dependent conformational changes. The present studies show that the structural consequences of each amino acid substitution in combinatorial mutant proteins were similar to those observed in the related single-mutant proteins, and therefore no synergistic effect between mutation sites was observed for this feature. With respect to protein stability, the effect of individual mutations can be understood from the structural changes observed for each. It is found that stability effects of the three mutation sites are independent and cumulative in multiple-mutant proteins. This reflects the independent nature of the structural changes induced at the three distally located mutation sites. In terms of heme reduction potential two effects are observed. For substitution of Phe82 by serine, the mechanism by which reduction potential is lowered is different from that occurring at either the Arg38 or the Asn52 site and is independent of residue replacements at these latter two positions. For Arg38 and Asn52, overlapping interactions lead to a higher reduction potential than expected from a strict additive effect of substitutions at these residues. This appears to arise from interaction of these two amino acids with a common heme element, namely, the heme propionate A group. The present results underscore the difficulty of predicting synergistic effects of multiple mutations within a protein.

Crystallography, X-Ray↗

PubMed2004

The epigenetic stability of the locus control region-deficient IgH locus in mouse hybridoma cells is a clonally varying, heritable feature.

Cis-acting elements such as enhancers and locus control regions (LCRs) prevent silencing of gene expression. We have shown previously that targeted deletion of an LCR in the immunoglobulin heavy-chain (IgH) locus creates conditions in which the immunoglobulin micro heavy chain gene can exist in either of two epigenetically inherited states, one in which micro expression is positive and one in which micro expression is negative, and that the positive and negative states are maintained by a cis-acting mechanism. As described here, the stability of these states, i.e., the propensity of a cell to switch from one state to the other, varied among subclones and was an inherited, clonal feature. A similar variation in stability was seen for IgH loci that both lacked and retained the matrix attachment regions associated with the LCR. Our analysis of cell hybrids formed by fusing cells in which the micro expression had different stabilities indicated that stability was also determined by a cis-acting feature of the IgH locus. Our results thus show that a single-copy gene in the same chromosomal location and in the presence of the same transcription factors can exist in many different states of expression.

Animals↗

PubMed2001

Leaving group stabilization by metal ion coordination and hydrogen bond donation is an evolutionarily conserved feature of group I introns.

To understand the behavior of group I introns on a biologically fundamental level, we must distinguish those traits that arise as the products of natural selection (selected traits) from those that arise as the products of neutral drift (non-selected traits). In practice, this distinction relies on comparing the similarities and differences among widely divergent introns to identify conserved traits. Here we address whether the strategies used by the eukaryotic group I intron from the Tetrahymena ciliate to stabilize the leaving group during splicing are maintained in the group I intron from the widely divergent Azoarcus bacterium. A substrate analogue containing a 3'-phosphorothiolate linkage, in which a sulfur atom replaces the bridging 3'-oxygen atom of the scissile phosphate, reacts 20-fold slower in the Azoarcus reaction than the corresponding unmodified substrate in the presence of Mg(II) as the only divalent cation. However, Mn(II) relieves this negative effect such that the 3'-S-P bond cleaves 21-fold faster than does the 3'O-P bond. Other thiophilic divalent metal ions such as Co(II), Cd(II), and Zn(II) similarly support cleavage of the S-P bond. These results indicate that a metal ion directly coordinates to the leaving group in the transition state of the Azoarcus ribozyme reaction. Additionally, the 3'-sulfur substitution eliminates the approximately 10(3)-fold contribution of the adjacent 2'-OH to transition state stabilization. Considering that sulfur accepts hydrogen bonds weakly compared to oxygen, this result suggests that the 2'-OH contributes to catalysis by donating a hydrogen bond to the 3'-oxygen leaving group in the transition state, presumably acting in conjunction with the metal ion to stabilize the developing negative charge. These same catalytic strategies of metal ion coordination and hydrogen bond donation operate in the Tetrahymena ribozyme reaction, suggesting that these features of catalysis have been conserved during evolution and thus extend to all group I introns. The two ribozymes also exhibit quantitative differences in their response to 3'-sulfur substitution. The Azoarcus ribozyme binds and cleaves the phosphorothiolate substrate more efficiently relative to the natural substrate than the Tetrahymena ribozyme under the same conditions, suggesting that the Azoarcus ribozyme better accommodates the phosphorothiolate at the active site both in the ground state and in the transition state. These differences may reflect either a less tightly knit Azoarcus structure and/or spatial deviations between backbone atoms in the two ribozymes that arise during divergent evolution, analogous to the well-documented relationship between protein sequence and structure.

Animals↗

PubMed2006

On the biomechanical stability of cementless straight conical hip stems.

The aim of the present study was to investigation in vitro the effect of deficient bone-implant contact on the primary stability of a straight conical stem. Various possible deficient contact patterns were derived from surgical simulations. The effect of stair climbing loads on the bone-implant micromotion was firstly investigated using a finite element model and then an in vitro test aimed at assessing primary stability. It was found that if the surface features are prevented from biting dense bone in a few small but critical regions, stem primary stability is completely lost. These results suggest that the surface features used in the axisymmetric stem under investigation can be too sensitive to deficient contact conditions, and thus should be augmented with additional antirotational fins. Preliminary tests showed that a stem with the addition of such fins presents good primary stability in all tested conditions.

Arthroplasty, Replacement, Hip↗

PubMed1997

Glycosylation beyond the Asn78-linked GlcNAc residue has a significant enhancing effect on the stability of the alpha subunit of human chorionic gonadotropin.

The effects of glycosylation beyond the Asn-linked GlcNAc residues on the stability of the alpha subunit of human chorionic gonadotropin are investigated, using enzymatic deglycosylation and NMR spectroscopy. Comparison of thermal denaturation profiles of both the intact alpha subunit and the alpha subunit carrying only GlcNAc monomers at both Asn52 and Asn78 established a small but significant decrease in thermal stability of the deglycosylated form. Since there is no secondary structure around Asn52 in the free subunit these results demonstrate that glycosylation beyond the Asn78-linked GlcNAc residue enhances the thermal stability of the alpha subunit of hCG. This feature has implications for understanding the effect of glycosylation on protein stabilization in general.

Acetylglucosamine↗

PubMed2004

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain.

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.

Animals↗

PubMed1996

Structural analysis of bovine somatotropin using monoclonal antibodies and the conformation-sensitive immunoassay.

Bovine somatotropin was studied with respect to thermal stability, quantitative thermal denaturation kinetics, and refolding potential following thermal denaturation using a panel of 6 monoclonal antibodies and the Conformation-Sensitive Immunoassay (CSI). The antibody panel consisted of 4 conformation-dependent and 2 sequence-specific antibodies. Each of the antibodies revealed unique thermal stability profiles for their respective epitopes suggesting that they each recognize different antigenic determinants. Comparing the thermal stability profiles generated with these antibodies allowed the stability of bovine somatotropin to be "dissected" based on individual structural features. The degree to which bovine somatotropin is stabilized by disulfide bonds was examined using CSI-based quantitative thermal denaturation kinetics profiles generated under reducing and nonreducing conditions. All of the conformational epitopes unfolded faster under reducing conditions indicating that the two disulfide bonds within the somatotropin molecule impart some degree of global stabilization. The ability of bovine somatotropin to refold after reducing or nonreducing thermal denaturation was also examined using the antibody panel and the CSI. The results show that, although significant refolding was evident for some epitopes, bovine somatotropin cannot refold to the native state following thermal denaturation under either reducing or nonreducing conditions.

Animals↗

PubMed1994

Thermodynamics of RNA folding in a conserved ribosomal RNA domain.

A small, 58 nt domain of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1108) is a highly conserved junction of three helices whose secondary structure has been established by phylogenetic comparisons. To detect any contributions of additional tertiary interactions, the thermal denaturation of the rRNA domain was followed by either UV hyperchromicity or calorimetry in buffers containing a wide range of Mg2+ concentrations. Several smaller fragments corresponding to two different hairpin stem-loop structures within the domain were also synthesized and melted for comparison with the larger molecule. A model of the secondary structure unfolding was devised, based on measured enthalpies and melting temperatures of the component hairpins and tabulated parameters of base-pair stacking and loop closure. The model closely simulates the observed melting data when three additional factors are included: two parameters to account for coaxial stackings within a junction of helices, and a set of undefined "tertiary" interactions that unfolds before the secondary structure and is preferentially stabilized by Mg2+. A critical feature of this model is a conserved pair, U1082/A1086, that is within the junction loop and hypothesized to stack with an adjacent helix. The model correctly predicts the effects of disrupting this pair in a U1086 sequence variant. Although the set of "tertiary" interactions contributes a significant fraction of the RNA unfolding enthalpy (delta H approximately 25 kcal/mol, out of 180 kcal/mol total), its overall stability is marginal at 37 degrees C.

Base Sequence↗