稳定的RNA发夹结构及其生物学功能

以UNCG、GNRA、CUUG(N=A、U、C或G,R=G或A)为端环能够形成稳定的、保守的发夹结构。高分辨率的溶液结构、晶体结构和计算机模拟等方法从原子水平上解析了这些发夹特殊的结构特征。在体内,它们发挥着重要的生物学功能:在折叠过程中作为折叠的起始位置帮助组织RNA分子正确折叠;与核酸受体结合参与三级相互作用;与蛋白质发生相互作用;阻止逆转录酶的延伸等等。另外,由于C(UUCG)G发夹极其稳定的特征,在体外RNA分子的实验测定中它还是稳定核酸结构的理想工具。这些稳定的发夹广泛分布于体内rRNA、催化RNA和非编码mRNA中。但在对人类编码区mRNA结构特征的研究当中,却未发现C(UUCG)G发夹。     

A test of the model to predict unusually stable RNA hairpin loop stability

To investigate the accuracy of a model [Giese et al., 1998, Biochemistry37:1094-1100 and Mathews et al., 1999, JMol Biol 288:911-940] that predicts the stability of RNA hairpin loops, optical melting studies were conducted on sets of hairpins previously determined to have unusually stable thermodynamic parameters. Included were the tetraloops GNRA and UNCG (where N is any nucleotide and R is a purine), hexaloops with UU first mismatches, and the hairpin loop of the iron responsive element, CAGUGC. The experimental values for the GNRA loops are in excellent agreement (deltaG degrees 37 within 0.2 kcal/mol and melting temperature (TM) within 4 degrees C) with the values predicted by the model. When the UNCG hairpin loops are treated as tetraloops, and a bonus of 0.8 kcal/mol included in the prediction to account for the extra stable first mismatch (UG), the measured and predicted values are also in good agreement (deltaG degrees 37 within 0.7 kcal/mol and TM within 3 degrees C). Six hairpins with unusually stable UU first mismatches also gave good agreement with the predictions (deltaG degrees 37 within 0.5 kcal/mol and TM within 8 degrees C), except for hairpins closed by wobble base pairs. For these hairpins, exclusion of the additional stabilization term for UU first mismatches improved the prediction (AG degrees 37 within 0.1 kcal/mol and TM within 3 degrees C). Hairpins with the iron-responsive element loop were not predicted well by the model, as measured deltaG degrees 37 values were at least 1 kcal/mol greater than predicted.     

Structural parameters affecting the kinetics of RNA hairpin formation

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program ‘Kinfold’. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem–loop structures.     

Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactions

RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical. We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE). This method was used herein to analyze a six-nucleotide RNA hairpin loop library. Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing. Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair. In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both. These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained. Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3. Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments. A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering). NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159. Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

The application of cluster analysis in the intercomparison of loop structures in RNA

We have developed a computational approach for the comparison and classification of RNA loop structures. Hairpin or interior loops identified in atomic resolution RNA structures were intercompared by conformational matching. The root-mean-square deviation (RMSD) values between all pairs of RNA fragments of interest, even if from different molecules, are calculated. Subsequently, cluster analysis is performed on the resulting matrix of RMSD distances using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis objectively reveals groups of folds that resemble one another. To demonstrate the utility of the approach, a comprehensive analysis of all the terminal hairpin tetraloops that have been observed in 15 RNA structures that have been determined by X-ray crystallography was undertaken. The method found major clusters corresponding to the well-known GNRA and UNCG types. In addition, two tetraloops with the unusual primary sequence UMAC (M is A or C) were successfully assigned to the GNRA cluster. Larger loop structures were also examined and the clustering results confirmed the occurrence of variations of the GNRA and UNCG tetraloops in these loops and provided a systematic means for locating them. Nineteen examples of larger loops that closely resemble either the GNRA or UNCG tetraloop were found in the large ribosomal RNAs. When the clustering approach was extended to include all structures in the SCOR database, novel relationships were detected including one between the ANYA motif and a less common folding of the GAAA tetraloop sequence.     

Co-conservation of rRNA tetraloop sequences and helix length suggests involvement of the tetraloops in higher-order interactions

Terminal loops containing four nucleotides (tetraloops) are common in structural RNAs, and they frequently conform to one of three sequence motifs, GNRA, UNCG, or CUUG. Here we compare available sequences and secondary structures for rRNAs from bacteria, and we show that helices capped by phylogenetically conserved GNRA loops display a strong tendency to be of conserved length. The simplest interpretation of this correlation is that the conserved GNRA loops are involved in higher-order interactions, intramolecular or intermolecular, resulting in a selective pressure for maintaining the lengths of these helices. A small number of conserved UNCG loops were also found to be associated with conserved length helices, consistent with the possibility that this type of tetraloop also takes part in higher-order interactions.     

Three-dimensional motifs from the SCOR, structural classification of RNA database: extruded strands, base triples, tetraloops and U-turns

Release 2.0.1 of the Structural Classification of RNA (SCOR) database, http://scor.lbl.gov, contains a classification of the internal and hairpin loops in a comprehensive collection of 497 NMR and X-ray RNA structures. This report discusses findings of the classification that have not been reported previously. The SCOR database contains multiple examples of a newly described RNA motif, the extruded helical single strand. Internal loop base triples are classified in SCOR according to their three-dimensional context. These internal loop triples contain several examples of a frequently found motif, the minor groove AGC triple. SCOR also presents the predominant and alternate conformations of hairpin loops, as shown in the most well represented tetraloops, with consensus sequences GNRA, UNCG and ANYA. The ubiquity of the GNRA hairpin turn motif is illustrated by its presence in complex internal loops.     

Isolation and characterization of thermodynamically stable and unstable RNA hairpins from a triloop combinatorial library

Hairpins are the most common elements of RNA secondary structure, playing important roles in RNA tertiary architecture and forming protein binding sites.Triloops are common in a variety of naturally occurring RNA hairpins, but little is known about their thermodynamic stability. Reported here are the sequences and thermodynamic parameters for a variety of stable and unstable triloop hairpins. Temperature gradient gel electrophoresis (TGGE) can be used to separate a simple RNA combinatorial library based on thermal stability [Bevilacqua, J. M., and Bevilacqua, P. C. (1998) Biochemistry 45, 15877-15884]. Here we introduce the application of TGGE to separating and analyzing a complex RNA combinatorial library based on thermal stability, using an RNA triloop library. Several rounds of in vitro selection of an RNA triloop library were carried out using TGGE, and preferences for exceptionally stable and unstable closing base pairs and loop sequences were identified. For stable hairpins, the most common closing base pair is CG, and U-rich loop sequences are preferred. Closing base pairs of GC and UA result in moderately stable hairpins when combined with a stable loop sequence. For unstable hairpins, the most common closing base pairs are AU and UG, and U-rich loop sequences are no longer preferred. In general, the contributions of the closing base pair and loop sequence to overall hairpin stability appear to be additive. Thermodynamic parameters for individual hairpins determined by UV melting are generally consistent with outcomes from selection experiments, with hairpins containing a CG closing base pair having a DeltaDeltaG degrees (37) 2.1-2.5 kcal/mol more favorable than hairpins with other closing base pairs. Sequences and thermodynamic rules for triloop hairpins should aid in RNA structure prediction and determination of whether naturally occurring triloop hairpins are thermodynamically stable.     

Effects of osmolytes on stable UUCG tetraloops and their preference for a CG closing base pair

Osmolytes have the potential to affect the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in the cell. To contribute to the understanding of the in vivo function of RNA we observed the effects of different classes of osmolytes on the UNCG tetraloop motif. UNCG tetraloops are the most common and stable of the RNA tetraloops and are nucleation sites for RNA folding. They also have a significant thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing UUCG loops was monitored using UV-Vis spectroscopy in the presence of osmolytes with different chemical properties. Interestingly, all of the osmolytes tested destabilized the hairpins, but all had little effect on the thermodynamic preference for a CG base pair, except for polyethylene glycol (PEG) 200. PEG 200 destabilized the loop with the CG closing base pair relative to the loop with a GC closing base pair. The destabilization was linear with increasing concentrations of PEG 200, and the slope of this relationship was not perturbed by changes in the hairpin stem outside of the closing pair. This result suggests that in the presence of PEG 200, the UUCG loop with a GC closing base pair may retain some preferential interactions with the cosolute that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes may influence how osmolytes tune the stability, and thus the function of a secondary structure motif in vivo.     

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.     

Evidence that folding of an RNA tetraloop hairpin is less cooperative than its DNA counterpart

Hairpin secondary structural elements play important roles in the folding and function of RNA and DNA molecules. Previous work from our lab on small DNA hairpin loop motifs, d(cGNAg) and d(cGNABg) (where B is C, G, or T), showed that folding is highly cooperative and obeys indirect coupling, consistent with a concerted transition. Herein, we investigate folding of the related, exceptionally stable RNA hairpin motif, r(cGNRAg) (where R is A or G). Previous NMR characterization identified a complex network of seven hydrogen bonds in this loop. We inserted three carbon (C3) spacers throughout the loop and found coupling between G1 of the loop and the CG closing base pair, similar to that found in DNA. These data support a GNRA motif being expandable at any position but before the G. Thermodynamic measurements of nucleotide-analogue-substituted oligonucleotides revealed pairwise-coupling free energies ranging from weak to strong. When coupling free energies were remeasured in the background of changes at a third site, they remained essentially unchanged even though all of the sites were coupled to each other. This type of coupling, referred to as "direct", is peculiar to the RNA loop. The data suggest that, for small stable loops, folding of RNA obeys a model with nearest-neighbor interactions, while folding of DNA follows a more concerted process in which the stabilizing interactions are linked through a conformational change. The lesser cooperativity in RNA loops may provide a more robust loop that can withstand mutations without a severe loss in stability. These differences may enhance the ability of RNA to evolve.     

Multinuclear NMR studies of DNA hairpins. 2. Sequence-dependent structural variations

The solution conformation of three related DNA hairpins, each with five bases in the loop, is investigated by proton and phosphorus 2D NMR methods. The sequences of the three oligomers are d(CGCGTTGTTCGCG), d(CGCGTTTGTCGCG), and d(CTGCTCTTGTTGAGCAG). One pair of hairpins shares the same stem sequence but differs in the loop, and the appearance of an unusual phosphate torsion in the stem is found to depend on the sequence in the loop of the hairpin. The second pair of hairpins shares the same loop region but differs in the stem sequence in that the base pair which closes the loop is a C-G or G-C pair. The pattern of NOEs reveals that the stacking arrangement in the loop region depends on the base pair that closes the stem. These results suggest that hairpin loop conformation and dynamics are sensitive to small changes in the loop and adjacent stem sequences. These findings are discussed in relation to sequence-dependent thermodynamic changes that have been observed in RNA hairpins.     

The effects of loop size on Sac7d-hairpin DNA interactions

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1-L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA.     

Analysis of melting transitions of the DNA hairpins formed from the oligomer sequences d[GGATAC(X)4GTATCC] (X = A, T, G, C)

Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3 degrees C for the T4 loop hairpin to 55.3 degrees C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop formation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions. Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.     

Understanding the thermodynamic stability of an RNA hairpin and its mutant.

The thermodynamic stability of RNA hairpin loops has been a subject of considerable interest in the recent past (Wimberly et al., 1991). There have been experimental reports indicating that the hairpins with a C(UUCG)G loop sequence are thermodynamically very stable (Wimberly et al., 1991). We used the solution structure of GGAC(UUCG)GUCC (Cheong et al., 1990; Varani et al., 1991) as the starting conformation in our attempt to understand its thermodynamic stability. We carried out molecular dynamics/free energy simulations to understand the basis for the destabilization of the C(UUCG)G loop by mutating cytosine (C7)-->uracil. Because of the limited length of simulation and the presence of kinetic barriers (solvent intervention) to the uracil-->cytosine mutation, all of our computed free energy differences are based on multiple forward simulations. Based on these calculations we find that the cytosine-->uracil mutation in the loop destabilizes it by approximately 1.5kcal/mol relative to that of the reference state, an A-form RNA but with cytosine (C7) looped out. This is the same sign and magnitude as that observed in the thermodynamic studies carried out by Varani et al.(1991). We have carried out free energy component analysis to understand the effect of mutating the cytosine residue to uracil on the thermodynamic stability of the C(UUCG)G hairpin loops. Our calculations show that the most significant contribution to the stability is from the phosphate group linking U5 and U6, which favors the cytosine residue over uracil by about 6.0 kcal/mol. The residues U5, U6, and G8 in the loop region also contribute significantly to the stability. The contributions from the salt and solvent compensate each other, indicating the dynamic nature of interactions of the environment with the nucleic acid system and the coupling between these two components.     

Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.