A hairpin structure is one of the most abundant secondary structural elements in RNA (ribonucleic acid), with RNA hairpins playing important structural and functional roles in providing nucleation sites for RNA folding, structural scaffolds and recognition sites for both proteins and nucleic acids. Tetraloops occur frequently in RNA molecules and account for approximately 55% of all hairpin loops in ribosomal RNA (rRNA) in Escherichia coli. The tetraloops in rRNA are highly constrained in sequence and approximately 70% of the tetraloop sequences belong to the UNCG or GNRA families, where N is any nucleotide and R is a purine. These frequently occurring tetraloops have higher thermal stability than the other four-nucleotide loops with the same stem. See also Nucleic Acids: General Properties, RNA Structure, and rRNA Structure
Many RNA tetraloops form compact and stable structures, which involve unusual basebase, basesugar, basephosphate and sugarphosphate interactions. In addition, base stacking extends to the loop region. UUCG, GCAA and CUUG tetraloops, for example, contain UG, GA and CG base pairs, respectively, leaving two highly constrained nucleotides in the loop. In fact, these loops should be considered mini-loops because they have only two loop nucleotides, but we will call them tetraloops for consistency. The structural characteristics of the tetraloops are summarized in Table 1. See also Base Pairing in RNA: Unusual Patterns, and RNA Structural Motifs
The sequence UNCG is frequently observed in RNA tetraloops and may serve as a nucleation site for RNA folding and as a protein-binding site. Hairpins containing UNCG tetraloops exhibit exceptionally high thermal stability. In the high-resolution nuclear magnetic resonance (NMR) structure of a UUCG hairpin (Cheong et al., 1990), unusual basebase, basesugar and basephosphate interactions are observed (Figure 1 and Figure 2a). The fourth G is in syn conformation. The 3¢-endo/syn conformation of the fourth G is further supported by Raman spectroscopy. Based on the identification of 2¢-OH of the first U and several nuclear Overhauser effects (NOEs), Allain and Varani (1995) have proposed a hydrogen bond between 2¢-OH of the first U and O6 of the fourth G (Figure 1g). The first and fourth bases interact with a bifurcated hydrogen bond between the first U carbonyl group and the imino and amino groups of the fourth G (Figure 1d). The existence of these hydrogen bonds are supported by the ultraviolet (UV) melting data of the UNCG tetraloops where the presence of 2¢-OH groups in the loop and NH
|Figure 2. UUCG (a) and GCAA (b) tetraloop structures. Hydrogen bonds are shown as dotted green lines. Carbon atoms are shown in green, phosphorus purple, nitrogen blue, oxygen red and hydrogen white.|
In addition to the hairpin structure solved by NMR, the UUCG-containing dodecamer 5¢GGACUUCGGUCC3¢ in a crystal assumes a double helical conformation containing two U·G wobble base pairs and two U·C mismatches. But even under the conditions used to grow the crystal, the RNA tetraloop maintained a single-strand hairpin conformation, which was monitored by
The GNRA hairpin loops are found frequently in catalytic, phage and signal-recognition particle (SRP) RNAs as well as in rRNAs. The GNRA and UNCG hairpin loops have been suggested to be evolutionarily interchangeable. The structure of GNRA (Jucker et al., 1996) shares many similarities with that of UNCG (Figure 2). The first G and fourth A form a sheared base pair (Figure 1e), leaving only a two-base loop like UNCG. Stacking of the bases in the loop extends to the third A in the 3¢ side. The GNRA hairpins change the direction of the phosphate backbone between the first and second nucleotides and this turn is stabilized by a basephosphate (Figure 1l) and ribosebase (Figure 1h) hydrogen bonds. All nucleotides in the stem and loop have anticonformation in contrast to UNCG, where the fourth G is in syn conformation. In a GCAA tetraloop, the second C is less ordered than the other nucleotides in the loop and is not stacked. But in a GAAA tetraloop, the second A seems to be stacked on the third A residue. The sugar conformation of the second and third nucleotides in a GNRA tetraloop exhibits dynamics between C2¢-endo and C3¢-endo.
The sheared G·A base pair (Figure 1e) is common in nature. The WatsonCrick faces of the guanine and adenine are available for tertiary contacts. The basetriplet interaction is one of the examples. Although the fourth A is involved in the basebase interaction, it has been suggested that the fourth A exhibits a chemical exchange process on the micro- to millisecond time scale. Jucker et al. (1996) have proposed a dynamic network of hydrogen bonds in this region of the tetraloop to explain the observed variations in the solution structures of several GNRA tetraloops. The dynamic model is consistent with T1 relaxation NMR data of the C8 atom of the fourth A. See also WatsonCrick Base Pairs and Nucleic Acids Stability
The solution structures of boxB RNA hairpins containing GACAA (Cai et al., 1998) and GAAGA (Legault et al., 1998) pentaloop, complexed with the transcriptional antitermination N-peptide of P22 and l, respectively, have been determined. Binding of the peptide in each case stabilizes the boxB RNA structure similar to that of the GNRA tetraloop. In the GACAA pentaloop of the boxB RNA hairpin, the first G and last A form the sheared G·A base pair (Figure 1e) which is stabilized by hydrogen bonds found in the GNRA loops. All the adenines in the loop are stacked and this stacking stabilizes the loop structure. The sugar conformation of the second A and third C is C2¢-endo. The third nucleotide in the GACAA is looped out to make an extensive hydrophobic interaction with the bound peptide. An arginine residue of the bound peptide forms hydrogen bonds with the guanine and with the adjacent backbone phosphate. The GAAGA pentaloop also forms a GNRA-like fold in which the fourth nucleotide is extruded from the loop, whereas the third nucleotide is looped out in the GACAA pentaloop. The fourth G adopts a syn glycosidic conformation and the base stacks on the ribose of the third A. It has been suggested that the GAAAA pentaloop also forms a GNRA-like conformation, since it can replace the GAAA tetraloop of the D5 domain in the group II intron without affecting the enzyme activity. See also Transcript Elongation and Termination in Bacteria
GNRA-like structures have also been found in the adenosine triphosphate (ATP)-binding aptamers. In the absence of adenosine monophosphate (AMP), the internal loop of the aptamer is largely unstructured. Upon binding of AMP, the aptamer adopts an L-shaped structure with two nearly orthogonal stems. In the AMPaptamer complex, the consecutive loop nucleotides GAA form a U turn (Figure 3) and interact with AMP to form a structure similar to that of the GNRA tetraloop where the AMP ligand functions as the fourth residue. The noncovalently attached AMP stacks between the third A and the base G of the stem. However, since the orientation of the AMP is parallel to the first G, the hydrogen-bonding scheme between AMP and the first G (Figure 1f) is different from the one observed in the sheared G·A base pair. See also Macromolecular Interactions: Aptamers
The CUUG tetraloop is frequently found in rRNAs. The first and fourth loop nucleotides form a standard WatsonCrick base pair and the second loop nucleotide interacts directly with the closing base pair of the stem by folding into the minor groove (Jucker and Pardi, 1995). The base plane of the first and fourth nucleotides is somewhat buckled although they are stacked on the stem bases. The position of the third U is not well-defined although it is partly stacked on the fourth G. The CUUG tetraloop is resistant to degradation promoted by Pb
The SL3 stemloop from the human immunodeficiency virus type 1 (HIV-1) packaging signal contains the GGAG tetraloop. NMR studies have shown that the GGAG tetraloop appears to be flexible, with the second G and fourth G bases extruded from the normal stacking arrangement (Pappalardo et al., 1998). The third A occupies a cavity large enough to jump rapidly between the stacking sites upon the first G and upon the 3¢-closing base G. The hydrogen-bonding loci of the second, third and fourth nucleotides are freely accessible. Upon nucleocapsid protein binding, the flexible RNA nucleotides become ordered. The first G stacks on the 5¢-stem base and forms a hydrogen bond with the fourth G. The second and third nucleotides are exposed and interact with the bound protein.
The last helix in bacterial 16S rRNA, helix 45, is capped by a methylated tetranucleotide loop, the sequence of which is m
The UGAA tetraloop, which is a conserved motif in eukaryotic 16S-like ribosomal RNA, displays a novel fold (Butcher et al., 1997a). The backbone turn occurs between the second G and third A in the loop. The syn orientation of the second G allows it to stack over the first U whereas the amino group makes a hydrogen bond to its own O5¢ phosphate oxygen (Figure 1n). The 2¢-OH of the second G lies within the hydrogen-bonding region of the phosphate oxygen of the third A (Figure 1j) and the third A is stacked over the fourth A. The first U and fourth A interact with each other. The amino group of the fourth A lies within the hydrogen-bonding distance of the O2, 2¢-OH and O4¢ groups of the first U. The unique fold of the UGAA tetraloop is stabilized by base stacking and a series of hydrogen bonds. See also Ribosomal RNA
The messenger RNA (mRNA) of bacteriophage T4 contains a hairpin structure with eight nucleotides in the loop. The hairpin plays an essential role in translational repression by binding to the T4 DNA (deoxyribonucleic acid) polymerase. The mutant CAAC tetraloop hairpin exhibits the same binding affinity for polymerase. Although the wild-type and the mutant hairpins have different secondary structures, the three-dimensional structures are very similar to each other (Mirmira and Tinoco, 1996). The tetraloop is stabilized by a hydrogen bond between the O2 of the first C and the amino group of the fourth C. The backbone turn occurs at the CpA step and the stacking extends to the second A on the 3¢-side. The WatsonCrick faces of the three nucleotides, AAC, in the tetraloop as well as in the wild-type octaloop are exposed to the solvent, providing a possible binding site for the polymerase (Mirmira and Tinoco, 1996). See also Bacteriophage T4
RNA folding is largely hierarchical; secondary structures, such as helices and loops, are formed whereas the tertiary structure is stabilized by long-range interactions. The 11-nt GAAA tetraloopreceptor is one of the best studied tertiary interactions. The tetraloopreceptors are commonly occurring motifs that have been found in the group I and group II self-splicing introns and RNase P, and they play critical roles in the folding of large RNAs. The structures of the free GAAA tetraloop and free receptor and tetraloopreceptor complex have been determined by X-ray crystallography (Battle and Doudna, 2002) and NMR spectroscopy (Butcher et al., 1997b; Davis et al., 2005).
The structure of the tetraloopreceptor interaction includes several motifs of RNA structures, such as A-minor interactions involving contacts between the tetraloop adenine nucleotides and the minor groove of a base pair that results in a triple base, an adenine platform (A-platform) in the receptor that forms stacking interactions with the 5¢-adenine of the tetraloop, ribose zipper motifs in which the 2¢-OH groups of the tetraloop and receptor form interdigitated hydrogen bonds, and a U turn in the GAAA tetraloop. Tetraloopreceptor interactions include 10 intermolecular hydrogen bonds and one base stacking interaction between the 5¢-adenine in the tetraloop and the A-platform.
The GAAA tetraloop docks to the receptor as a relatively rigid body. The tetraloop binds to the receptor by stacking on an A-platform and hydrogen bonding to a complementary minor groove pocket forming A-minor interactions and ribose zippers. However, the receptor undergoes relatively large conformational changes on tetraloop binding. The unbound receptor is an A-zipper with cross-strand stacking of three adenines and a noncanonical base pair. In the bound receptor, two adenines form an intrastrand A-platform that stacks on the 5¢-adenine of the tetraloop, and a uridine is bulged out. Mg
The GANC tetraloop is found within the ¢ interaction of group IIC introns (Toor et al., 2008). The backbone of the GANC tetraloop is a U turn-like conformation, as in the GNRN tetraloop. The sugar pucker in the first guanosine of GANC is a 2¢-endo, whereas a 3¢-endo sugar pucker is present in GNRA. The first G is pushed towards a minor groove and the remaining three bases stack on the closing base pair. Unusually, the GANC tetraloopreceptor interaction consists of only a base stack between the third N base of the tetraloop and a bulged purine of the receptor and does not contain an A-platform. The GANC tetraloopreceptor interaction in group IIC introns contributes to proper orientation, rather than energetic stabilization (Keating et al., 2008).
The MS2 coat protein binds to the translational operator stem-loop of the RNA bacteriophage MS2, which contains an AUUA tetraloop and acts as a translational repressor. In solution, the RNA exists as an ensemble of differently base-paired/base-stacked states at equilibrium (Borer et al., 1995). The complex structure shows sequence-specific interactions between conserved RNA bases and protein residues (Valegârd et al., 1997). The RNA interacts with the protein through phosphate groups, ribose and nucleotide bases. The third U and fourth A nucleotides form an extensive hydrogen bond network with the protein. The third U stacks onto the tyrosine side chain of the protein.
Yeast RNase III (Rnt1p) is a double-stranded RNA (dsRNA) endonuclease. Rnt1p cleavage sites are at stem-loops that are capped by a terminal conserved AGNN tetraloop located within 1316 base pairs of the cleavage sites. The AGNN tetraloops enhance Rnt1p-binding and are required for Rnt1p processing (Chanfreau et al., 2000). Rnt1p recognizes the fold of the AGNN tetraloop rather than the conserved sequence (Wu et al., 2004). The solution structure of Rnt1p and the AGNN tetraloop revealed that the N-terminal helix fits into the minor groove of the RNA tetraloop and the top of the stem by nonsequence-specific contacts with the sugarphosphate backbone and the two nonconserved tetraloop nucleotides. The enzyme acts as a molecular ruler that recognizes the AGNN tetraloop and cleaves the RNA substrate at a fixed distance to the recognition site.
The sarcin/ricin loop (SRL) is a highly conserved sequence found in the RNA of all large ribosomal subunits and is crucial for ribosomal functions such as initiation, elongation and termination of ribosome-directed protein synthesis. The SRL RNA is a critical component of the binding site for EFs (embryonic fibroblasts) and possibly for other GTPase (guanosine triphosphatase) protein factors such as IF2. The SRL RNA structures contain a bulged G-motif and a GAGA tetraloop (Correll et al., 1998). The site-specific, toxic sarcin and ricin families bind, and covalently modify, the SRL and inactivate ribosomes leading to cell death. Ricin recognizes only the GAGA tetraloop and depurinates the first A. The glycosidic bond of the first A flips to a syn from an anticonformation upon ricin binding (Yang et al., 2001). The third nucleotide in the GAGA loop is also important for ricin recognition, since ricin does not recognize the structurally similar GAAA tetraloop. Sarcin primarily recognizes the bulged G-motif located a fixed (~11 Å) distance away from the scissible bond (Correll et al., 2004). Sarcin binds SRL RNA nonspecifically, followed by recognition of the SRL structure of the bulged G-motif and GAGA tetraloop (Korennykh et al., 2007). Upon binding sarcin, the GAGA tetraloop undergoes conformational change to position the nucleophile for an in-line attack on the scissible bond. The structure of ricin-SRL also supports the molecular ruler model. See also Base Flipping, and ProteinRNA Interactions
Basephosphate interactions are found in the UNCG, GNRA and UGAA tetraloops. The hydrogen bonds between the amino group of the bases and the phosphate oxygen seem to stabilize the sharp turn in the backbone of these tetraloops.
In the UUCG tetraloop, NOEs from the third C aromatic to the first and second U sugar protons define a close contact between the cytosine exocyclic amino and the phosphate oxygen connecting the first and second nucleotides (Allain and Varani, 1995; Figure 1k). This hydrogen bond has been identified from the cytosine amino resonances by using
The U turn motif (Figure 3) found in the anticodon and TC loops of transfer RNA (tRNA) is observed in the catalytic core of the hammerhead ribozyme, GNRA tetraloops (Jucker et al., 1996), the SRL loop (Correll et al., 1998) and the ATP-binding RNA aptamer. The three-base U turn motif (G/UNR) is characterized by a turn in the backbone after the first nucleotide, which is stabilized by ribosebase and basephosphate hydrogen bonds (Figure 3). Two hydrogen bonds, between 2¢-OH of G/U and N7 of R (Figure 1h) and amino/imino groups of G/U and N7 and 3¢-phosphate of R (Figure 1l), stabilize the conformation. Two different hydrogen-bonding schemes have been proposed for the basephosphate interaction of the GNRA tetraloop (Figure 1l and m). After the backbone turn, the next two bases are stacked over each other and the stacking continues to the 3¢-stem in the case of tetraloops. See also Transfer RNA, and Transfer RNA Structure
In the UGAA tetraloop, the second G in syn conformation is stacked over the first U. A potential hydrogen bond from 2¢-OH of the second G to the phosphate oxygen of the third A has been suggested (Figure 1j). The backbone turn occurs between the second and third nucleotides and is stabilized by a basephosphate interaction between the third A amino group and the second G phosphate (Figure 1n).
Stacking of the Third Base
The third base stacking in the tetraloops is a common feature. All the tetraloop structures solved so far exhibit the third base stacking although the stacking partners of the third base are different. The functional and structural importance of this character should be studied further. In the UNCG tetraloop, the third C stacks on the U·G base pair. For the GGAG tetraloop, the third base jumps rapidly between stacking on the first G and the 3¢-stem. For the rest of the tetraloops (GNRA, CUUG, m
The basebase interactions between the first and the fourth nucleotides are found in most of the tetraloops (UNCG, GNRA, CUUG, UGAA and CAAC). The tetraloops having these interactions should be considered as mini-loops having only two loop nucleotides. For these tetraloops, buckling between the first and the fourth bases is usually observed. Both of the two-base nucleotides have one or more C2¢-endo sugar conformation to help bridge the stem. For the specific hydrogen-bonding schemes of the basebase interactions, refer to the section on Structure and Figure 1d.
One or both of the two loop nucleotides of the two-base loops often exhibit structural dynamics. The position of the second nucleotide in the UNCG and GNRA loops is not well-defined compared to the other loop nucleotides. The sugar conformation of all the loop nucleotides in the UGAA loop shows dynamics. In the CUUG tetraloop, the second U is well-defined by the NOE data and interacts with the loop C·G and the first stem base pairs in the minor groove. The position of the third U is not well-defined although it lies in the major groove. The unstructured nucleotide in the two-base loops may be important for recognition and RNAprotein interactions.
- A ligand-binding oligonucleotide which has been isolated by an in vitro selection technique.
- Bifurcated hydrogen bond
- A hydrogen bond where a single hydrogen bond acceptor or donor is involved in two hydrogen bonds.
- Molecular dynamics
- Methods that evaluate the force field energy and its analytical derivatives and move all atoms using Newton's laws of motion.
- NOE (Nuclear Overhauser effect)
- A change in the integrated nuclear magnetic resonance signal intensity of a nuclear spin when another spin is saturated.
- A process whereby nonradiative energy transfer takes place from an excited spin to the lattice degrees of freedom of the system.
- WatsonCrick face
- The side of a base or a base pair having the hydrogen bond donors and acceptors that participate in WatsonCrick base pair.