Experimental and computational studies of the G[UUCG]C RNA tetraloop

In prokaryotic ribosomal RNAs, most UUCG tetraloops are closed by a C-G base-pair. However, this preference is greatly reduced in eukaryotic rRNA species where many UUCG tetraloops are closed by G-C base-pairs. Here, biophysical properties of the C[UUCG]G and G[UUCG]C tetraloops are compared, using experimental and computational methods. Thermal denaturation experiments are used to derive thermodynamic parameters for the wild-type G[UUCG]C tetraloop and variants containing single deoxy substitutions in the loop. A comparison with analogous experiments on the C[UUCG]G motif shows that the two RNA species exhibit similar patterns in response to the substitutions, suggesting that their loop structures are similar. This conclusion is supported by NMR data that suggest that the essential UUCG loop structure is maintained in both tetraloops. However, NMR results show that the G[UUCG]C loop structure is disrupted prior to melting of the stem; this behavior is in contrast to the two-state behavior of the C[UUCG]G molecule. Stochastic dynamics simulations using the GB/SA continuum solvation model, run as a function of temperature, show rare conformational transitions in several G[UUCG]C simulations. These results lead to the conclusion that substitution of a G-C for a C-G closing base-pair increases the intrinsic flexibility of the UUCG loop.     

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.     

Highly sampled tetranucleotide and tetraloop motifs enable evaluation of common RNA force fields

Recent modifications and improvements to standard nucleic acid force fields have attempted to fix problems and issues that have been observed as longer timescale simulations have become routine. Although previous work has shown the ability to fold the UUCG stem–loop structure, until now no group has attempted to quantify the performance of current force fields using highly converged structural populations of the tetraloop conformational ensemble. In this study, we report the use of multiple independent sets of multidimensional replica exchange molecular dynamics (M-REMD) simulations with different initial conditions to generate well-converged conformational ensembles for the tetranucleotides r(GACC) and r(CCCC), as well as the larger UUCG tetraloop motif. By generating what is to our knowledge the most complete RNA structure ensembles reported to date for these systems, we remove the coupling between force field errors and errors due to incomplete sampling, providing a comprehensive comparison between current top-performing MD force fields for RNA. Of the RNA force fields tested in this study, none demonstrate the ability to correctly identify the most thermodynamically stable structure for all three systems. We discuss the deficiencies present in each potential function and suggest areas where improvements can be made. The results imply that although “short” (nsec-μsec timescale) simulations may stay close to their respective experimental structures and may well reproduce experimental observables, inevitably the current force fields will populate alternative incorrect structures that are more stable than those observed via experiment.     

Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis

The HIV-1 transactivation response element (TAR) RNA binds a variety of proteins and is a target for developing anti-HIV therapies. TAR has two primary binding sites: a UCU bulge and a CUGGGA apical loop. We used NMR residual dipolar couplings, carbon spin relaxation (R₁ and R₂), and relaxation dispersion (R_1ρ) in conjunction with molecular dynamics and mutagenesis to characterize the dynamics of the TAR apical loop and investigate previously proposed long-range interactions with the distant bulge. Replacement of the wild-type apical loop with a UUCG loop did not significantly affect the structural dynamics at the bulge, indicating that the apical loop and the bulge act largely as independent dynamical recognition centers. The apical loop undergoes complex dynamics at multiple timescales that are likely important for adaptive recognition: U31 and G33 undergo limited motions, G32 is highly flexible at picosecond-nanosecond timescales, and G34 and C30 form a dynamic Watson-Crick basepair in which G34 and A35 undergo a slow (∼30 μs) likely concerted looping in and out motion, with A35 also undergoing large amplitude motions at picosecond-nanosecond timescales. Our study highlights the power of combining NMR, molecular dynamics, and mutagenesis in characterizing RNA dynamics.     

Solution Structure of IseA,an Inhibitor Protein of dl-Endopeptidases from Bacillus subtilis,Reveals a Novel Fold with a Characteristic Inhibitory Loop

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, dl-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the dl-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation dl-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3₁₀-helix, and eight β-strands, which is a novel fold like a “hacksaw.” Noteworthy is a dynamic loop between β4 and the 3₁₀-helix, which resembles a “blade.” The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4–3₁₀ loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4–3₁₀ loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.     

Molecular dynamics simulations of RNA kissing-loop motifs reveal structural dynamics and formation of cation-binding pockets

Explicit solvent molecular dynamics (MD) simulations were carried out for three RNA kissing–loop complexes. The theoretical structure of two base pairs (2 bp) complex of H3 stem–loop of Moloney murine leukemia virus agrees with the NMR structure with modest violations of few NMR restraints comparable to violations present in the NMR structure. In contrast to the NMR structure, however, MD shows relaxed intermolecular G-C base pairs. The core region of the kissing complex forms a cation-binding pocket with highly negative electrostatic potential. The pocket shows nanosecond-scale breathing motions coupled with oscillations of the whole molecule. Additional simulations were carried out for 6 bp kissing complexes of the DIS HIV-1 subtypes A and B. The simulated structures agree well with the X-ray data. The subtype B forms a novel four-base stack of bulged-out adenines. Both 6 bp kissing complexes have extended cation-binding pockets in their central parts. While the pocket of subtype A interacts with two hexacoordinated Mg²⁺ ions and one sodium ion, pocket of subtype B is filled with a string of three delocalized Na⁺ ions with residency times of individual cations 1–2 ns. The 6 bp complexes show breathing motions of the cation-binding pockets and loop major grooves.     

Thermodynamics of 2'-ribose substitutions in UUCG tetraloops

The ribose 2'-hydroxyl group confers upon RNA many unique molecular properties. To better appreciate its contribution to structure and stability and to monitor how substitutions of the 2' hydroxyl can alter an RNA molecule, each loop pyrimidine ribonucleotide in the UUCG tetraloop was substituted with a nucleotide containing either a fluorine (2'-F), hydrogen (2'-H), amino (2'-NH2), or methoxy (2'-OCH3) group, in the context of both the C:G and G:C loop-closing base pair. The thermodynamic parameters of these tetraloop variants have been determined and NMR experiments used to monitor the structural changes resulting from the substitutions. The modified riboses are better tolerated in the G[UUCG]C tetraloop, which may be due to its increased loop flexibility relative to the C[UUCG]G loop. Even for these simple substitutions, the free-energy change reflects a complex interplay of hydrogen bonding, solvation effects, and intrinsic pucker preferences of the nucleotides.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

We report on the three dimensional structure of an RNA hairpin containing a 2',5'-linked tetraribonucleotide loop, namely, 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U(2'p5')U(2'p5')C(2'p5')G(2'p5')). We show that the 2',5'-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar-base contacts, all of which contribute to the extra stability of this hairpin structure.     

Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence

Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA polyadenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3′-ends of most of the mRNA molecules in bacteria are characterized by a stem–loop structure. We show here that in vitro PAP I activity is inhibited by a stem–loop structure. A tail of two to six nucleotides located 3′ to the stem–loop structure is sufficient to overcome this inhibition. These results suggest that the stem–loop structure located in most of the mRNA 3′-ends may function as an inhibitor of polyadenylation and degradation of the corresponding RNA molecule. However, RNA 3′-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.     

Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.     

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

Extra stable 2',5'-linked RNA loops

We prepared hairpins that differ in the connectivity of phosphodiester linkages in the loop (RNA vs 2', 5'-RNA). We find that the stability of the extra stable RNA hairpin 5'-rGGAC(UUCG)GUCC-3' is the same as that observed for the hairpin containing a 2',5'RNA loop, i.e. 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U2'p5'U2'p5' C2'p5'G2'p5'). Also significant is the finding that when the stem is duplex DNA, duplex 2',5'-RNA, or DNA:2',5'-RNA, hairpins with the UUCG loop are more stable than those with UUCG loop.     

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。     

Molecular dynamics reveals the stabilizing role of loop closing residues in kissing interactions: comparison between TAR-TAR* and TAR-aptamer

A RNA aptamer (R06) raised against the trans- activation responsive (TAR) element of HIV-1 was previously shown to generate a loop–loop complex whose stability is strongly dependent on the selected G and A residues closing the aptamer loop. The rationally designed TAR* RNA hairpin with a loop sequence fully complementary to the TAR element, closed by U,A residues, also engages in a loop–loop association with TAR, but with a lower stability compared with the TAR–R06 complex. UV absorption monitored thermal denaturation showed that TAR–TAR*(GA), in which the U,A kissing residues were exchanged for G,A, is as stable as the selected TAR–R06 complex. Consequently, we used the TAR–TAR* structure deduced from NMR studies to model the TAR–R06 complex with either GA, CA or UA loop closing residues. The results of the molecular dynamics trajectories correlate well with the thermal denaturation experiments and show that the increased stability of the GA variant results from an optimized stacking of the bases at the stem–loop junction and from stable interbackbone hydrogen bonds.     

Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase

Solution NMR spin relaxation experiments and classical MD simulations are used to study the dynamics of triosephosphate isomerase (TIM) in complex with glycerol 3-phosphate (G3P). Three regions in TIM exhibit conformational transitions on the micros-ms time scale as detected by chemical exchange broadening effects in NMR spectroscopy: residue Lys 84 on helix C, located at the dimeric interface; active site loop 6; and helix G. The results indicate that the conformational exchange process affecting the residues of loop 6 is the correlated opening and closing of the loop. Distinct processes are responsible for the chemical exchange linebroadening observed in the other regions of TIM. MD simulations confirm that motions of individual residues within the active site loop are correlated and suggest that the chemical exchange processes observed for residues in helix G arise from transitions between 3(10)- and alpha-helical structures. The results of the joint NMR and MD study provide global insight into the role of conformational dynamic processes in the function of TIM.