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.     

Domain rearrangement of SRP protein Ffh upon binding 4.5S RNA and the SRP receptor FtsY

The signal recognition particle (SRP) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. In Escherichia coli, SRP consists of 4.5S RNA and a protein, Ffh, that recognizes the signal peptide emerging from the ribosome and the SRP receptor at the membrane, FtsY. In the present work, we studied the interactions between the NG and M domains in Ffh and their rearrangements upon complex formation with 4.5S RNA and/or FtsY. In free Ffh, the NG and M domains are facing one another in an orientation that allows cross-linking between positions 231 in the G domain and 377 in the M domain. There are binding interactions between the two domains, as the isolated domains form a strong complex. The interdomain contacts are disrupted upon binding of Ffh to 4.5S RNA, consuming a part of the total binding energy of 4.5S RNA-Ffh association that is roughly equivalent to the free energy of domain binding to each other. In the SRP particle, the NG domain binds to 4.5S RNA in a region adjacent to the binding site of the M domain. Ffh binding to FtsY also requires a reorientation of NG and M domains. These results suggest that in free Ffh, the binding sites for 4.5S RNA and FtsY are occluded by strong domain-domain interactions which must be disrupted for the formation of SRP or the Ffh-FtsY complex.     

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA.

Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.     

NMR structure of the 3' stem-loop from human U4 snRNA

The NMR structure of the 3' stem-loop (3'SL) from human U4 snRNA was determined to gain insight into the structural basis for conservation of this stem-loop sequence from vertebrates. 3'SL sequences from human, rat, mouse and chicken U4 snRNA each consist of a 7 bp stem capped by a UACG tetraloop. No high resolution structure has previously been reported for a UACG tetraloop. The UACG tetraloop portion of the 3'SL was especially well defined by the NMR data, with a total of 92 NOE-derived restraints (about 15 per residue), including 48 inter-residue restraints (about 8 per residue) for the tetraloop and closing C-G base pair. Distance restraints were derived from NOESY spectra using MARDIGRAS with random error analysis. Refinement of the 20mer RNA hairpin structure was carried out using the programs DYANA and miniCarlo. In the UACG tetraloop, U and G formed a base pair stabilized by two hydrogen bonds, one between the 2'-hydroxyl proton of U and carbonyl oxygen of G, another between the imino proton of G and carbonyl oxygen O2 of U. In addition, the amino group of C formed a hydrogen bond with the phosphate oxygen of A. G adopted a syn orientation about the glycosidic bond, while the sugar puckers of A and C were either C2'-endo or flexible. The conformation of the UACG tetraloop was, overall, similar to that previously reported for UUCG tetraloops, another member of the UNCG class of tetraloops. The presence of an A, rather than a U, at the variable position, however, presents a distinct surface for interaction of the 3'SL tetraloop with either RNA or protein residues that may stabilize interactions important for active spliceosome formation. Such tertiary interactions may explain the conservation of the UACG tetraloop motif in 3'SL sequences from U4 snRNA in vertebrates.     

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.     

Role for both DNA and RNA in GTP hydrolysis by the Neisseria gonorrhoeae signal recognition particle receptor

The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that P(pilE) DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.     

A conserved major groove antideterminant for Saccharomyces cerevisiae RNase III recognition

Rnt1p, the only known Saccharomyces cerevisiae RNase III double-stranded RNA endonuclease, plays important roles in the processing of precursors of ribosomal RNAs and small nuclear and nucleolar RNAs and in the surveillance of unspliced pre-mRNAs. Specificity of cleavage by Rnt1p relies on the presence of RNA tetraloop structures with the consensus sequence AGNN at the top of the target dsRNA. The sequences of 79 fungal RNase III substrates were inspected to identify additional conserved sequence elements or antideterminants that may contribute to Rnt1p recognition of the double-stranded RNA. Surprisingly, U-A sequences at the base pair adjacent to the conserved terminal tetraloop (closing base pair) were found to be absent from all but one inspected sequence. Analysis of chemically modified variants of the closing base pair showed that the presence of exocyclic groups in the major groove of purines 3' to the last nucleotide of the tetraloop inhibits Rnt1p cleavage without strongly inhibiting Rnt1p binding. We propose that these groups interfere with the recognition of the RNA substrate by the catalytic domain of Rnt1p. These results identify exocyclic groups of purines in the major groove downstream of the tetraloop as a major antideterminant in S. cerevisiae RNase III activity, and suggest a rationale for their apparent counter selection in RNA processing sites.     

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.     

Synergistic actions between the SRP RNA and translating ribosome allow efficient delivery of the correct cargos during cotranslational protein targeting

During cotranslational protein targeting by the Signal Recognition Particle (SRP), the correct cargo accelerates stable complex assembly between the SRP and SRP receptor (FtsY) by several orders of magnitude, thus enabling rapid and faithful cargo delivery to the target membrane. The molecular mechanism underlying this cargo-induced rate acceleration has been unclear. Here we show that the SRP RNA allows assembly of the SRP-FtsY complex to be specifically stimulated by a correct cargo, and, reciprocally, a correct cargo enables the SRP RNA to optimize its electrostatic interactions with FtsY. These results combined with recent structural work led us to suggest a "conformational selection" model that explains the synergistic action of the SRP RNA with the cargo in accelerating complex assembly. In addition to its previously proposed role in preventing the premature dissociation of SRP and FtsY, we found that the SRP RNA also plays an active role in ensuring the formation of productive assembly intermediates, thus guiding the SRP and FtsY through the most efficient pathway of assembly.     

Crystal structure of the ffh and EF-G binding sites in the conserved domain IV of Escherichia coli 4.5S RNA

BACKGROUND: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptide-containing proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation. RESULTS: We have determined by multiple anomalous dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment containing binding sites for both Ffh and EF-G. This fragment consists of three helices connected by a symmetric and an asymmetric internal loop. In contrast to NMR-derived structures reported previously, the symmetric loop is entirely constituted by non-canonical base pairs. These pairs continuously stack and project unusual sets of hydrogen-bond donors and acceptors into the shallow minor groove. The structure can therefore be regarded as two double helical rods hinged by the asymmetric loop that protrudes from one strand. CONCLUSIONS: Based on our crystal structure and results of chemical protection experiments reported previously, we predicted that Ffh binds to the minor groove of the symmetric loop. An identical decanucleotide sequence is found in the EF-G binding sites of both 4.5S RNA and 23S rRNA. The decanucleotide structure in the 4.5S RNA and the ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA might interact with EF-G and function in translating ribosomes.     

Dynamics and stability of GCAA tetraloops with 2-aminopurine and purine substitutions

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G-->AP) and the third (A-->PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A-->PUR) or in the C-G base pair closing the tetraloop (G-->AP) to some extent influenced the loop structure and dynamics. These loops did not display the long-lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the glycosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

Role of SRP RNA in the GTPase cycles of Ffh and FtsY.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.     

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.     

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.     

Dynamics and Stability of GCAA Tetraloops with 2-Aminopurine and Purine Substitutions

Abstract

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G→AP) and the third (A→PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A→PUR) or in the C-G base pair closing the tetraloop (G→AP) to some extent influenced the loop structure and dynamics. These loops did not display the long- lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the gly- cosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP

In cotranslational preprotein targeting in Escherichia coli, the signal recognition particle (SRP) binds to the signal peptide emerging from the ribosome and, subsequently, interacts with the signal recognition particle receptor, FtsY, at the plasma membrane. Both FtsY and the protein moiety of the signal recognition particle, Ffh, are GTPases, and GTP is required for the formation of the SRP-FtsY complex. We have studied the binding of GTP/GDP to FtsY as well as the SRP-FtsY complex formation by monitoring the fluorescence of tryptophan 343 in the I box of mutant FtsY. Thermodynamic and kinetic parameters of the FtsY complexes with GDP, GTP, and signal recognition particle are reported. Upon SRP-FtsY complex formation in the presence of GTP, the fluorescence of tryptophan 343 increased by 50 % and was blue-shifted by 10 nm. We conclude that GTP-dependent SRP-FtsY complex formation leads to an extensive conformational change in the I box insertion in the effector region of FtsY.     

Identification and characterization of Streptococcus pneumoniae Ffh, a homologue of SRP54 subunit of mammalian signal recognition particle

Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.     

Base dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin relaxation: correlations with structure and stability.

Intramolecular dynamics of guanine and uracil bases in a 14-nt RNA hairpin including the extraordinarily stable UUCG tetraloop were studied by 15N spin relaxation experiments that are sensitive to structural fluctuations occurring on a time scale of picoseconds to nanoseconds. The relaxation data were interpreted in the framework of the anisotropic model-free formalism, using assumed values for the chemical shift anisotropies of the 15N spins. The rotational diffusion tensor was determined to be symmetric with an axial ratio of 1.34 +/- 0.12, in agreement with estimates based on the ratio of the principal moments of the inertia tensor. The model-free results indicate that the bases of the G x U pair in the tetraloop are at least as rigid as the interior base pairs in the stem, whereas the 5'-terminal guanine is more flexible. The observed range of order parameters corresponds to base fluctuations of 19-22 degrees about the chi torsion angle. The results reveal dynamical consequences of the unusual structural features in the UUCG tetraloop and offer insights into the configurational entropy of hairpin formation.     

Extending the family of UNCG-like tetraloop motifs: NMR structure of a CACG tetraloop from coxsackievirus B3

Stable RNA tetraloop motifs are found frequently in biologically active RNAs. These motifs carry out a wide variety of functions in RNA folding, in RNA-RNA and RNA-protein interactions. A great deal of knowledge about the structures and functions of tetraloop motifs has accumulated largely due to intensive theoretical, biochemical, and biophysical studies on three most frequently occurring families of tetraloop sequences, namely, the cUNCGg, the cGNRAg, and the gCUUGc sequences. Our knowledge surely is not exhaustive, and efforts are still being made to gain a better understanding. Here we report the NMR structure of a uCACGg tetraloop that occurs naturally within the cloverleaf RNA structure of the 5'-UTR of coxsackievirus B3. This tetraloop is the major determinant for interaction between the cloverleaf RNA and viral 3C protease, which is an essential part of a ribonucleoprotein complex that plays a critical role in the regulation of viral translation and replication. Our structure shows that the CACG tetraloop is closed by a wobble U.G base pair. The structure of the CACG tetraloop is stabilized by extensive base stacking and hydrogen bonding interactions strikingly similar to those previously reported for the cUUCGg tetraloop. Identification of these hallmark structural features strongly supports the existence of an extended YNCG tetraloop family. The U.G base pair closing the stem and the A residue in the loop introduce some small structural and themodynamic distinctions from the canonical cUUCGg tetraloop that may be important for recognition by the viral 3C protease.