Anomalous hairpin formation in an oligodeoxyribonucleotide.

An accurate method for deriving molar absorptivity-temperature profiles applied to a set of single-stranded oligodeoxyribonucleotides shows that the undecamer CGAGTTTGACGp exists in a hairpin conformation involving Watson-Crick base pairing between the two terminal CG dinucleotides. The hairpin, which has a transition midpoint of 40 degrees C in 0.115 M Na+, is unusually stable in comparison with previously reported hairpins. A non-linear least squares analysis of the undecamer's profile in terms of a two-state equilibrium model indicates that the hairpin-to-coil transition occurs with an enthalpy change about twice that expected if only combinations of Watson-Crick base-paired stacking interactions are considered. The analogous hairpin structure (containing an identical CG/CG stem) assignable to the complementary strand CGTCAAACTCGp does not form above 0 degrees C. Measurements on the two undecamers indicate that variation in non Watson-Crick interactions within the loops of two similar hairpins can produce a difference in stability of at least 2.2 kcal/mol (25 degrees C, 0.115 M Na+), roughly equal to the amount contributed to a double helix by a 5'-CG-3'/5'-CG-3' base-paired stacking interaction.     

Correlation of RNA Secondary Structure Statistics with Thermodynamic Stability and Applications to Folding

The accurate prediction of the secondary and tertiary structure of an RNA with different folding algorithms is dependent on several factors, including the energy functions. However, an RNA higher-order structure cannot be predicted accurately from its sequence based on a limited set of energy parameters. The inter- and intramolecular forces between this RNA and other small molecules and macromolecules, in addition to other factors in the cell such as pH, ionic strength, and temperature, influence the complex dynamics associated with transition of a single stranded RNA to its secondary and tertiary structure. Since all of the factors that affect the formation of an RNAs 3D structure cannot be determined experimentally, statistically derived potential energy has been used in the prediction of protein structure. In the current work, we evaluate the statistical free energy of various secondary structure motifs, including base-pair stacks, hairpin loops, and internal loops, using their statistical frequency obtained from the comparative analysis of more than 50,000 RNA sequences stored in the RNA Comparative Analysis Database (rCAD) at the Comparative RNA Web (CRW) Site. Statistical energy was computed from the structural statistics for several datasets. While the statistical energy for a base-pair stack correlates with experimentally derived free energy values, suggesting a Boltzmann-like distribution, variation is observed between different molecules and their location on the phylogenetic tree of life. Our statistical energy values calculated for several structural elements were utilized in the Mfold RNA-folding algorithm. The combined statistical energy values for base-pair stacks, hairpins and internal loop flanks result in a significant improvement in the accuracy of secondary structure prediction; the hairpin flanks contribute the most.     

The effect of sodium ion concentration on intrastrand base-pairing in single-stranded DNA.

The salt-induced formation of duplex structure (primarily hairpin loops) in denatured calf thymus DNA was monitored by measuring the decrease in absorbance at 260 nm as a function of increasing sodium ion concentration. It was found that this process was noncooperative and could be accurately described by the mass-action expression for the reversible formation of a binary complex: single strand (coil) + free sodium ion <==> hairpin (with associated sodium ion). The equilibrium constant for the transition was found to be 6 (M Na+)-1. The extrapolated absorbance at infinite salt concentration represents 11% hyperchromicity, which is one third of the hyperchromicity of denatured DNA in the absence of salt (36%).     

The folding of the hairpin ribozyme: dependence on the loops and the junction

In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form.     

Metal ion binding and the folding of the hairpin ribozyme

The hairpin ribozyme comprises two formally unpaired loops carried on two arms of a four-way helical RNA junction. Addition of divalent metal ions brings about a conformational transition into an antiparallel structure in which there is an intimate association between the loops to generate the active form of the ribozyme. In this study, we have used fluorescence resonance energy transfer to analyze the global folding of the complete ribozyme, and the simple four-way junction derived from it, over a wide concentration range of divalent and monovalent metal ions. The simple junction undergoes an ion-induced rotation into an antiparallel form. In the presence of a constant background concentration of sodium ions, the magnesium-ion-induced transition is characterized by noncooperative binding with a Hill coefficient n = 1. By contrast, the magnesium-ion-induced folding of the complete ribozyme is more complex, involving two distinct binding phases. The first phase occurs in the micromolar range, and involves the cooperative binding of at least three magnesium ions. This can also be achieved by high concentrations of sodium ions, and is therefore likely to be due to diffuse binding of cations at the junction and the interface of the loop-loop interaction. The second phase occurs in the millimolar range, and can only be induced by divalent metal ions. This transition occurs in response to the noncooperative, site-specific binding of magnesium ions. We observe a good correlation between the extent of ion-induced folding and cleavage activity.     

DNA hairpins: fuel for autonomous DNA devices

We present a study of the hybridization of complementary DNA hairpin loops, with particular reference to their use as fuel for autonomous DNA devices. The rate of spontaneous hybridization between complementary hairpins can be reduced by increasing the neck length or decreasing the loop length. Hairpins with larger loops rapidly form long-lived kissed complexes. Hairpin loops may be opened by strand displacement using an opening strand that contains the same sequence as half of the neck and a "toehold" complementary to a single-stranded domain adjacent to the neck. We find loop opening via an external toehold to be 10-100 times faster than via an internal toehold. We measure rates of loop opening by opening strands that are at least 1000 times faster than the spontaneous interaction between hairpins. We discuss suitable choices for loop, neck, and toehold length for hairpin loops to be used as fuel for autonomous DNA devices.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.     

Some hydrodynamic and optical properties of polyribonucleotides

The size and shape of four polyribonucleotides were studied by sedimentation and viscosity measurements. The results were correlated with their conformations based on optical activity studies. Polyriboadenylic acid in acidic solution dimerizes into two double-stranded forms, one with half-protonated bases and the other fully protonated. The fully protonated form is somewhat less asymmetrical than the half-protonated form. Polyriboguanylic acid in alkaline solution (near the second pK_a of guanine) undergoes a time-dependent disaggregation and the final form shows little base stacking. In acidic solution, it demonstrates a reversible transition near the first pK_a of guanine but without evidence of disaggregation. Polyribocytidylic acid undergoes a transition upon half protonation of the bases, but its molecular weight remains unchanged with pH. The results suggest that this polymer assumes a hairpin structure in acidic solution. Polyribouridylic acid has some degree of base stacking: at room temperature. A transition to a hairpin structure occurs at low temperature.     

Circular dichroism measurements show that C.C+ base pairs can coexist with A.T base pairs between antiparallel strands of an oligodeoxynucleotide double-helix.

We have studied the coil-to-helix transition of the DNA oligomer d(C4A4T4C4), using circular dichroism measurements to monitor the formation of A.T base pairs within the central self-complementary A4T4 region and the formation of protonated C.C+ base pairs at the ends of the oligomer. We found that both A.T and C.C+ base pairs formed in a coordinated fashion as the temperature and pH were lowered. The CD data of the helix form of the oligomer were consistent with the presence of paired oligomers, but not with hairpin loops. The pKa for formation of C.C+ base pairs between the C4 ends of the oligomer was higher than the pKa for formation of C.C+ base pairs in d(C8), indicating that the formation of C.C+ base pairs in the oligomer was influenced by the presence of a paired A4T4 region. We conclude that A.T and C.C+ base pairs coexist in the self-complex of the oligomer and, therefore, that C.C+ base pairs can form between antiparallel DNA strands.     

The Effects of Trinucleotide Repeats Found in Human Inherited Disorders on Palindrome Inviability in Escherichia Coli Suggest Hairpin Folding Preferences in Vivo

Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that are associated with several human inherited disorders. We present evidence consistent with the folding of these trinucleotide repeats into hairpin loops at the center of a long DNA palindrome in vivo. Our assay utilizes a palindrome in bacteriophage λ, the center of which determines its ability to inhibit plaque formation in a manner that is consistent with folding into a hairpin or cruciform structure. We show that central inserts of even numbers of d(CAG)·d(CTG) repeats inhibit plaque formation more than do odd numbers. Both d(CAG)(2)·d(CTG)(2) and d(CGG)(2)·d(CCG)(2) central sequences behave like DNA sequences known to form two-base loops in vitro, suggesting that they may also form compact and stable loops. By contrast, repeats of d(GAC)·d(GTC) do not show any evidence consistent with unusual loop stability. These results agree with in vitro evidence that the unstable repeats can form hairpin secondary structures and suggest a favored position of folding. We discuss the potential roles of secondary structures, DNA replication and recombination in models of repeat tract expansion.     

Loopstructures in synthetic oligodeoxynucleotides.

A comparative nuclear magnetic resonance study of the hydrogen-bonded imino protons in a series of synthetic DNA fragments is presented. The fragments ATCCTA(Tn)TAGGAT are in principle capable of forming either a self-complementary hairpin loop structure (monomer form) or an interior loop structure (dimeric form). It has been shown, that for n = 1 only the dimer structure is present in aqueous solution, whereas the exclusive existence of the hairpin loop structure is indicated for n = 3, 4 & 5. Surprisingly, for n = 2 two different structures appear to be present in solution. Concentration studies show that both monomers and dimers exist side by side in this case. Hairpins as well as interior loops form extra "melting sites" in addition to the wellknown fraying phenomenon at the terminus of the double helix.     

A minimal RNA promoter for minus-strand RNA synthesis by the brome mosaic virus polymerase complex

The approximately 150 nt tRNA-like structure present at the 3' end of each of the brome mosaic virus (BMV) genomic RNAs is sufficient to direct minus-strand RNA synthesis. RNAs containing mutations in the tRNA-like structure that decrease minus-strand synthesis were tested for their ability to interact with RdRp (RNA-dependent RNA polymerase) using a template competition assay. Mutations that are predicted to disrupt the pseudoknot and stem B1 do not affect the ability of the tRNA-like structure to interact with RdRp. Similarly, the +1 and +2 nucleotides are not required for stable template-RdRp interaction. Mutations in the bulge and hairpin loops of stem C decreased the ability of the tRNA-like structure to interact with RdRp. Furthermore, in the absence of the rest of the BMV tRNA, stem C is able to interact with RdRp. The addition of an accessible initiation sequence containing ACCA3' to stem C created an RNA capable of directing RNA synthesis. Synthesis from this minimal minus-strand template is dependent on sequences in the hairpin and bulged loops.     

Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.     

Dynamic Monte Carlo simulations of globular protein folding/unfolding pathways. II. Alpha-helical motifs

Dynamic Monte Carlo simulations of the folding pathways of alpha-helical protein motifs have been undertaken in the context of a diamond lattice model of globular proteins. The first question addressed in the nature of the assembly process of an alpha-helical hairpin. While the hairpin could, in principle, be formed via the diffusion-collision-adhesion of isolated performed helices, this is not the dominant mechanism of assembly found in the simulations. Rather, the helices that form native hairpins are constructed on-site, with folding initiating at or near the turn in almost all cases. Next, the folding/unfolding pathways of four-helix bundles having tight bends and one and two long loops in the native state are explored. Once again, an on-site construction mechanism of folding obtains, with a hairpin forming first, followed by the formation of a three-helix bundle, and finally the fourth helix of the native bundle assembles. Unfolding is essentially the reverse of folding. A simplified analytic theory is developed that reproduces the equilibrium folding transitions obtained from the simulations remarkably well and, for the dominant folding pathway, correctly identifies the intermediates seen in the simulations. The analytic theory provides the free energy along the reaction co-ordinate and identifies the transition state for all three motifs as being quite close to the native state, with three of the four helices assembled, and approximately one turn of the fourth helix in place. The transition state is separated from the native conformation by a free-energy barrier of mainly energetic origin and from the denatured state by a barrier of mainly entropic origin. The general features of the folding pathway seen in all variants of the model four-helix bundles are similar to those observed in the folding of beta-barrel, Greek key proteins; this suggests that many of the qualitative aspects of folding are invariant to the particular native state topology and secondary structure.     

Antisense RNA control of gene expression in bacteriophage P22. II. Kinetic mechanism and cation specificity of the pairing reaction.

The bacteriophage P22 sar RNA-ant mRNA pairing reaction was characterized kinetically. The pairing reaction proceeds by a three-step pathway. First, reversible base pairs form between complementary hairpin loops in sar RNA and ant RNA (Kd = 270 nM). Next, stable duplex formation initiates between single-stranded nucleotides in sar RNA and ant RNA; the ant RNA nucleotides are at the bottom of a hairpin stem that is partially accessible. Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin then occurs, to form a partially paired intermediate (k2 = 12 min(-1). Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin (k3 = 7 min(-1). Experiments with sar RNA sequence and length variants demonstrate that the precise structures of both sar RNA hairpins affect the kinetic parameters. The pairing reaction is Mg2+-dependent, and shows high specificity for the required cation. Maximal pairing rates are achieved when more than one Mg2+ ion is bound. The cation-dependence and specificity indicate a requirement for Mg2+-dependent tertiary structure.     

Protonation of deoxycytidine residues in dC4 tetraloops: UV spectrophotometric study of dC10 and d(A14C4T14)

It is shown that component analysis could be applied to study the UV difference spectra of cytidine oligomers and hairpin oligonucleotides with cytidines in the loop region in order to account for the melting and titration results in terms of cytidine stacking and protonation. Upon acid titration, the dC(10) oligomer undergoes cooperative conformational transition at pH 6.3 accompanied by protonation and formation of the i-structure with half of the residues protonated. The stability of the hemiprotonated structure increases with decreasing pH, the i-structure persisting still in the region of pH相似文献   

Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins

The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.     

Poliovirus snapback double-stranded RNA isolated from infected HeLa cells is deficient in poly(A).

A portion of poliovirus double-stranded RNA (25 to 50%) isolated from infected HeLa cells contains hairpin loops at one end of the duplex structure. These structures rapidly reformed double-stranded molecules after denaturation and appeared as molecules of up to two times genome length upon electrophoresis in denaturing agarose gels. A second form of poliovirus double-stranded RNA was readily denaturable into genome length strands. When the hairpin RNA was treated with S1 nuclease, subsequent denaturation resulted in formation of strands of up to genome length. Hairpin molecules contained very little, if any, poly(A) sequences, suggesting that the hairpin forms after nucleolytic removal of the 3' end of plus-strand templates. We conclude that the hairpin double-stranded RNA found in infected cells is likely generated by intracellular nicking and self-priming and that it does not represent an intermediate in the process of RNA replication.     

Stretching single-stranded DNA: interplay of electrostatic, base-pairing, and base-pair stacking interactions.

Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.     

A GCUA tetranucleotide loop found in the poliovirus oriL by in vivo SELEX (un)expectedly forms a YNMG-like structure: Extending the YNMG family with GYYA

The cloverleaf structure in the 5'-untranslated region of enterovirus RNA that regulates viral RNA replication contains an evolutionarily conserved YNMG tetraloop closed by a Y-G base pair. This loop is believed to interact specifically with the viral protease 3C. To further characterize the specificity of this interaction, the tetraloop and two flanking base pairs of the poliovirus RNA were randomized, and viable viral clones were obtained using in vivo SELEX. Among many different mutants with the canonical YNMG sequences to be described elsewhere, a large-plaque-forming clone contained a deviating uGCUAg sequence. The NMR structure of a small hairpin capped with uGCUAg that we present here shows that the GCUA tetraloop adopts a novel fold, which is highly similar to that of the YNMG tetraloop with common stacking properties and hydrogen-bond interactions including an unusual syn conformation of the adenosine. Thermodynamic studies show moderate stabilities of hairpins with canonical YNMG and the novel GCUA loops, which, together with the similarity of spatial structures, illustrates that the tetraloop structure itself is crucial for the RNA-protein interaction required for the viral replication. A re-evaluation of the ribosomal secondary structure database reveals a hairpin containing a GCUA loop, which covaries with YNMG and is involved in a tertiary interaction, and in the 50S ribosomal subunit from Haloarcula marismortui the structurally comparable apex of stem-loop 35a is a recognition site for protein L2. These observations show a more general occurrence and importance of the so-far unrecognized GYYA hairpin loops.