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RNA folding
RNA folding Anna Marie Pyle and Justin B Green C o l u m b i a University, N e w York, U S A The number of known motifs for RNA folding and RNA tertiary organization is expanding rapidly as we learn more about the diverse biological functions of RNA. Problems in protein and RNA folding have melded in recent investigations of ribonucleoprotein folding. Theoretical and experimental models are rapidly being developed for the pathways and stabilizing forces involved in RNA folding. Current Opinion in Structural Biology 1995, 5:303-310
Introduction In spite of its deceptively simple primary structure, R N A carries out a broad spectrum of biological functions beyond its informational role in gene expression. We now know that catalytic R N A molecules (ribozymes) are capable of catalyzing numerous chemical transformations. A large number of R N A structural motifs are now being recognized to be important in regulating diverse biochemical processes, including the translation of particular genes and the expression of viral genomes. Consequently, it is important for us to expand our understanding of R N A folding. In this review, we detail work in the past year on the building blocks for folding, and follow it with a section on how these units are assembled. The effects of metals and base modifications on P,.NA folding are assessed. New information on the folds of particular RNAs is provided, together with a section on the important new topic ofribonucleoprotein folding. All of this is concluded with a review of new information on the pathways and order of events involved in R N A folding.
Architectural elements of RNA folding: defining the building blocks The arrangement of R.NA structural elements in three-dimensional space is achieved by a process called R N A tertiary folding, The most fundamental structural elements as far as folding of R N A is concerned are the helical regions of secondary structure, which have stabilities that can be calculated with reasonable accuracy [1]. Considerable effort is being expended on defining additional units of P.NA folding. These motif~ are often stable enough to have biological functions of their own, but they may also function within the context of larger RNAs. They include particular kinds of pseudoknots,
loops that cap helices, loops within helices, regions of R N A mispairing, nucleoside triple interactions [2], quadruplexes and U-turns. Pseudoknots are interlocked regions of coaxially stacked helices that are commonly involved in R N A binding and folding. A pseudoknot structure proposed for regions of bacteriophage (I)29 prohead R N A is involved in DNA packaging [3]. Pseudoknotting is a motif with a high capacity for molecular recognition, as shown by the large number of pseudoknot structures derived from in vitro selection schemes. For example, an R N A with a high affinity for cyanocobalamin is proposed to have a pseudoknot structure [4°]. This interaction is particularly dependent on lithium ions, which are so small and unshielded that they can allow tight packing of phosphate residues within the folded RNA. An unusual motif in rat retrotransposon VL30 I:(NA was also proposed to fold specifically in the presence of Li +, so the effect of this ion may be general [5]. The P,.ev responsive element (tkR~) of HIV has an open internal loop of R N A that undergoes a conformational change upon binding of the Rev protein. In the complex, the Ioop region forms an underwound helix made up of purine-purine pairs that dramatically widen the major groove of the R N A [6"',7°']. This wide major groove of unusual accessibility may be a general feature imposed by tracts of G--G and G-A pairs imbedded within a helix. Although base functional groups in the major groove are not exposed in normal RNA helices, the R R E motif may be one strategy for increasing the accessibility of the numerous functional groups located m the major groove of R N A helices. Gautheret et al. [8"] observed that phylogenetically conserved G-A mismatches occur singly or in patterns of two tandem G - A mismatches. They proposed that by exposing purine functional groups to the solvent, these G--A motifs ,nay facilitate R N A tertiary interactions or protein binding [8°].
Abbreviations FRET~fluorescence resonance energy transfer, HDV--hepatitis delta virus; RRE--Rev responsiveelement,
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Nucleicacids Another group of internal loops that constitute a distinct and ubiquitous R N A folding motifs include structures variously termed Loop B (hairpin ribozyme), Loop E (eukaryotic 5S RNA) and the sarcin/ricin loop (16S RNA). The sequence of this asymmetrical loop motif is highly conserved [9"]. UV irradiation of the hairpin ribozyme results in a highly efficient cross-link across the loop B region [10"]. This result, together with those from modification interference studies, shows that the motif has a distinctive tertiary architecture [9°]. Electrophoretic phasing experiments on the symmetric Escherichia coli 5S R N A Loop E demonstrated the presence of a rigid bend within an overall helical structure [11"]. Despite the bending and the overwinding of the helix by 27 °, none of the bases were pushed out of the helix and the structure remained stacked.
Assembly of motifs into tertiary structures It is often necessary for individual blocks of R N A secondary structure to condense into tertiary structure. The folding of structural elements takes place through the formation of long-range tertiary interactions, which are now being defined in terms of discrete molecular patterns of chemical bonding. The backbone substituents that line the outside of R N A helices are important points of tertiary contact. In particular, ribose U-hydroxyls can bond directly to a variety of substitutents, setting a helical element into place within large R N A structures. This type of bonding arrangement appears to be particularly significant when the interactions mediated by T-hydroxyls occur in groups, as shown in a recent examination of tertiary interactions between the substrate recognition helix and the core of the Tetrahymena ribozyme ([12"]; see also Wahl and Sundaralingam, pp 282-295 and Tuschl et al., pp 296-302, this issue). Because all ribose residues have a U-hydroxyl group, additional elements may be required to control the specificity of U-hydroxyl interactions within the tertiary structure. In the case of the Tetrahymena ribozyme, a conserved G - U wobble pair is situated at the ribozyme cleavage site, immediately adjacent to an array of important U-hydroxyls. When this G--U pair is replaced with a G--C pair, the 2'-hydroxyl groups no longer dock into place and the enzymatic behavior of the ribozyme is significantly disrupted [13°%14°°]. The coaxial stacking of helices can fundamentally direct the overall fold of an RNA. The sequence dependence of coaxial stacking was recently analyzed and observed to follow the same nearest-neighbor rules that govern the formation of contiguous helices [15°]. For example, if there are three helices arranged in space with similar intervening constraints, one would predict that the two that come together in a coaxial stack may have terminal base pairs with the highest favorable fi:ee energy for
stacking, such as two G - C pairs, rather than a G - C pair and an A - U pair. The P4-P6 coaxial stacking interaction has a dominant effect on the overall fold and dimensions of the Tetrahymena ribozyme [16"'] (Fig. 1). A striking long-range tertiary interaction, involving specific binding of G N R A tetraloops (where N is any nucleotide and R is any purine) to G--C pairs within helical regions, was recently identified in a wide variety of different R N A molecules. Modification interference analysis on the Tetrahymena ribozyme and deletion mutants of it suggested that a tertiary interaction takes place between the GAAA tetraloop of P5abc (Fig. 1) and a C - G pair in region P6a [17"]. In the td group I intron, mutational and kinetic analysis showed that bases 3 and 4 of a conserved GUGA tetraloop specifically recognize nucleotide functional groups in the minor groove of two stacked A - U and G - C base pairs [18"°]. The tetraloop-helix docking motif was visualized at high resolution during crystallographic analysis of the hammerhead ribozyme (Fig. 2). Although the ribozyme itself does not contain it, the motif was formed as a consequence of intermolecular interactions between two ribozyme molecules. In this case, the principal interactions occured between GAAA tetraloop bases and two stacked G--C pairs on another molecule [19"']. Certain types of hairpin loops can bind to each other, as shown by N M R studies of a "kissing hairpin" [20°]. Circular permutation analysis of RNase P and domains of the Tetrahymena ribozyme reveals that the position of 5'- and 3'-ends in a correctly folded and functionally active R N A can be somewhat arbitrary [16"',21"]. This implies that location of the ends is not necessarily an element of importance in tertiary folding.
Trans domain analysis provides a way to quantitate the effects of tertiary interactions and folding of large R N A molecules. Once the independent folding domains of an R N A are determined, they may be transcribed as separate pieces that associate solely through tertiary interactions. If the R N A has catalytic activity, kinetics and binding studies can be used to evaluate the interaction energies and mechanistic role of specific folding domains. The mechanistic role of catalytic domain 5 (D5) of the group II intron was evaluated in this way. Despite the apparent lack of canonical base-pairing interactions between D5 and other components of the intron, D5 was observed to bind strongly to them, with a Kd of approximately 300 nM [22"]. A kinetic framework for D5 reactivity was also established that facilitates dissection ofsubstituent effects on either tertiary interaction energy or chemistry. Similarly, separation of RNase P into two domains facilitated investigation of metal binding and folding energetics in that molecule [21°]. The P4-P6 domain of the Tetrahymena ribozyme is an independently folding domain that can also be provided in trans to remaining portions of the molecule [17"]. Its contribution to ribozyme folding was monitored by modification interference and electron microscopy.
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The role of metal ions and base modifications in folding Metal ions play a critical role in folding and catalysis by R N A , although their effects have remained difficult to define because metals contribute to R N A stabilization in several ways. These distinct functions have been recently clarified through thermal denaturation experiments on different types of R N A molecules [23°']. Laing et al. [23 °°] show that if metal ions are binding an R N A non-specifically, thus playing a role in charge screening and duplex formation, a plot of the reciprocal of the melting temperature of the R N A (Tm-I) against [Mg2+] will be sigmoidal. If metal ions bind an R N A site-specifically, this plot will be linear. R N A molecules with a high degree of tertiary structure show this linear dependence. In one R N A , linear behavior was specific for Mg 2+, whereas in another, Co(NH3)63+ could substitute for Mg2+. This shows that there are
Fig. 1. Secondary structural representation of the Tetrahymena intron (upper case letters) and flanking exon sequences (lower case letters). The P1 duplex Iocatecl in the middle of this schematic contains the 5"-splice site or cleavage site, marked by an arrow at the conserved G - U wobble pair. The second arrow (near Pg.0) indicates the position of the 3'-splice site. Flanking the PI duplex are folded core elements, including the P4-P6 domain to the left and the P3-P7 domain to the right. The P5abc domain is shown to the left of P4-P6, with which it interacts. Thick lines represent continuity of the strands, and no nucleotides have been omitted. Arrowheads indicate the 5'--)3' polarity of the strands. Base pairings are represented by dark dashes. The light dashes extending from some of the bases indicate positions of known tertiary contact. Reproduced from [53] with permission.
at least three general roles for metals in R N A folding: non-specific binding; specific Mg2+ coordination; and binding to high-affinity sites through charge density or outer-sphere coordination rather than through the formation of direct metal contacts. In vivo, most functional R N A s are modified posttranscriptionally with a variety of functional groups. These are now known to have important effects on R N A folding. N M R studies comparing unmodified with modified tRNA^la show that base modifications strengthen metal-binding sites [24°]. A recent crystal structure of unmodified tRNAPhe shows that it is missing a water molecule near the nucleotide normally occupied by a W-base [25]. This suggests that modifications may influence the organization of water and thereby influence macromolecular stability.
An interesting report about modifications that reflects generally on the problem o f R N A folding involves
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Fig. 2. The long-range tertiary interaction between a GAAA tetraloop and a tandem G~C pair determined by crystallography. (a) A schematic of major stem interactions between two molecules of the hammerhead ribozyme in the unit cell. (b) A high-resolution illustration of the interactions of loop 4 adenosine (AL4 , on molecule 1 in [a]) with the GT4-CT4 base pair (on molecule 2). Note the abundance of base-backbone interactions and the additional intraloop contacts within molecule 1, between G U and AL4. Reproduced from [33 °°] with permission.
tRNA from hyperthermophilic bacteria [26°°]. As the temperature o f bacterial culture medium was raised, t R N A became modified more extensively. Three modifications known to promote greater helical rigidity (and in some cases better stacking) became especially prevalent. The modifications helped stabilize secondary structure. Interestingly, once tertiary structure was formed in these variously modified RNAs, they all had similar melting temperatures. This suggests that the modifications may be important along a folding pathway, rather than in maintenance o f folded structure.
Folding of specific RNAs A number o f recent papers have provided important information on the conformation and structure o f specific R N A molecules, particularly the smaller ribozymes and tRNA. Linkers were inserted between domains [27], and chemical modification studies were conducted [9°] in order to probe the overall folding of
the hairpin ribozyme and the dependence o f the process on Mg 2+. High-temperature analysis of the structure of genomic hepatitis delta virus (HDV) ribozyme resulted in greater sensitivity of mutational analysis and facilitated development of more refined structural models [28]. Chemical probing [29] and deletional studies on forms o f HDV ribozyme [30] revealed additional information about HDV folding and stability. Molecular modeling studies provided new proposals for the folding o f unusual mitochondrial tRNAs [31] and for the construction of three-dimensional models of large RNAs [32°]. Some of the most detailed information available on R N A folding comes from the first crystal structure of a ribozyme, reported in 1994. Before determination of the structure o f the hammerhead ribozyme [33°°], our only detailed view of folded R N A came from crystal structures o f tRNA. In the hammerhead structure, several core nucleotides form a set of G - A pairs that stack onto stem II of the ribozyme (Fig. 3). This structure is coaxially stacked with stem III. The ribozyme then forms a roughly Y-shaped structure as stem I curves alongside stem II and forms a bed of core nucleotides in the junction between the stern I and stem II helices. The unpaired core nucleotides form a defined R N A motif known as the "U-turn", which is identical in conformation to the uridine turn in the anticodon of t R N A Phe. The relative orientation o f the hammerhead stems was also examined using transient electric birefringence measurements [34°]. Fluorescence resonance energy transfer (FRET) measurements combined with molecular modeling provided an additional detailed structure of the hammerhead ribozyme [35°°]. Although the helical orientations and global conformation determined by F R E T are similar to those in the crystal structure, the conformation of core nucleotides differs. There are salient differences between the molecules used in the two studies that may have affected their overall structures. A recent cross-linking study provided evidence for multiple tertiary conformations of the hammerhead ribozyme [36°], and this structural variability may explain differences between recent models of the ribozyme. The conformation and folding of larger RNAs was also the subject o f many investigations in the past year. Results from chemical protection studies, phylogenetic analyses, mutagenesis and kinetics were combined to produce a working model of RNase P tertiary structure [37°]. Results from photoaffinity cross-linking o f circularly permuted RNase P was also used to generate a second structural model [38°]. Although the models appear superficially different, they share many common features and differ only in the relative orientations of certain subdomains of the molecule. Phylogeny and mutagenesis studies revealed a long-range tertiary interaction across the large loop o f R N a s e P [39], a feature that may help to further constrain the structure. Conformational variability of domains within the group II intron was explored through a combination o f kinetic and thermal denaturation experiments [40]. The folding
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Fig. 3. The three-dimensional structure of the hammerhead ribozyme, determined by X-ray crystallography. A ribbon diagram of the structure illustrates the major architectural features of the ribozyme, including the stack of stem It on stem 111,and the catalytic core of the ribozyme in the valley between stems I and II. Note the position of the uridine turn in the lower right of the structure and the tandem G-A mismatches stacked between stems II and III. Reproduced from [33°'] with permission.
of group I intron domains was visualized directly using electron microscopy of the Tetrahymena ribozyme. This study provided direct proof o f the presence of a sharp kink believed to exist between two rigid portions of the P5 domain [41°'].
Ribonucleoprotein folding Although the intrinsic properties of R N A molecules enable them to act as catalysts and units of molecular recognition, most of them function together with proteins in vivo. Given the intimate cooperation that exists between many R N A s and proteins, it may be increasingly important to consider a more unified concept in macromolecular organization: ribonucleoprotein folding. As the term implies, certain R N A s and proteins may fold together, making it important to unite concepts in R N A and protein folding. Specific classes of ribonucleoprotein folding have been demonstrated in recent examples from the literature and a recent review describes known motifs for RNA-protein interaction [42% The Tetrahymena ribozyme contains a large, independently folding domain known as P5abc, which can participate in the folding and activation of the ribozyme even when added in trans (Fig. 1). It was shown that this same function can also be provided by a protein known as Cyt-18, a tyrosyl t R N A synthetase [43"°]. The protein specifically binds and activates the
ribozyme only if it is missing the P5abc domain. This work conclusively demonstrates that certain RNAs and proteins can perform the same roles in stabilizing the active tertiary fold of an R N A molecule. The interaction of U1A protein with an R N A hairpin provides an example of an R N A molecule that folds only in the presence of a protein. In this example of induced fit, N M R and crystallographic studies reveal that a loop sequence in the R N A undergoes a massive conformational change [44°'], binding to the surface of an exposed J-sheet through many hydrogen-bonding contacts and base-stacking with a tyrosyl side chain [44°',45°']. In this example, the protein structure changes little upon R N A binding. Recent studies of t R N A lle suggest that certain R N A tertiary interactions are actually broken and new ones are formed upon synthetase binding [46]. A very different story is revealed in biochemical analyses of R e v - R R E binding. This RNA-protein interaction may best be described as a 'co-folding', in which association results in radical conformational changes in both R N A and protein [47°°]. Detailed kinetic analyses reveal that nucleocapsid protein can function as a chaperone for R N A folding [48°°]. The protein has effects on duplex annealing, stimulating product dissociation rate from the hammerhead ribozyme and thereby increasing the multiple-turnover rate of reaction (kcat). Nucleocapsid protein acts as a folding chaperone by eliminating a 'misfolded trap', or less-active conformation, that can be adopted by the ribozyme-substrate complex. This demonstrates that even non-specific RNA-binding proteins can function as R N A chaperones.
Pathways for RNA folding Now that motifs for local and tertiary folding of R N A have been described, we can attempt to explain how an R N A molecule gets to the folded state. R N A folding is believed to be distinct from protein folding in that R N A secondary structural elements are very stable and capable of forming independently of tertiary structure. This has led to the concept that R N A secondary structure forms rapidly and precedes the packaging of R N A into tertiary structure. Recent work shows that this idea needs to be extended in new directions and that different RNAs will not necessarily follow this canonical pathway. An important modification of RNA-folding theory is the need to recognize a hierarchy of individual RNA-folding domains. For example, in the Tetrahymena ribozyme, folding of the catalytic core is dependent upon formation of the independent P4-P6 domain [49°]. The folding pathway for the ribozyme was dissected using time-resolved oligonucleotide probe hybridization [50°°]. This study showed that the P4--P6 domain forms first, but that the rate-limiting step in overall folding is
308
Nucleicacids assembly of the P3-P7 domain. Once P3--P7 is folded, global tertiary organization rapidly follows. Thus, the rate-limiting step in this folding pathway is the formation of a domain, rather than tertiary organization of the final state. It is not yet clear whether formation of secondary or tertiary structures actually limits the rate of P3-P7 folding. In a model for the unfolding of a domain in the R,NA of the large ribosomal subunit [51•'], the pathway is dissected into five sequential transitions. The first transition is the unfolding of the magnesium-dependent tertiary structure, which occurs before the unfolding of secondary structure. The other transitions represent the coupled unfolding of four different helical units. Although this example appears to follow the standard paradigm for R N A folding, it is interesting that the secondary structural transitions are linked to formation of a specific pairing interaction in the junction loop between helices. The notion that I~NA tertiary structure always folds last is challenged by studies on the unfolding pathway of a complex pseudoknot [52°°]. In that case, the tertiary structure was not easily separated from the secondary structure and it is proposed that tertiary interactions may be required for the formation of a secondary structure element that was unstable on its own.
Conclusion The motifs and thermodynamic basis for R N A folding received considerable attention during the past year. This is partly because of an abundance of new structural information. The first crystal structure of a large R N A molecule other than tlLNA was reported [33°°]. This structural investigation of the hammerhead ribozyme provided a new window into overall R N A architecture and also revealed high-resolution information on longrange tertiary interactions. Numerous NMI:( studies revealed dramatic conformational changes in R N A and, together with biochemical and crystallographic studies, set the stage for new thinking on ribonucleoprotein folding. Throughout the year, spectroscopic and biochemical studies continued to elucidate the motifs that are fundamental to R N A folding, showing how they coalesce and interact to shape the tertiary structure of an R N A molecule.
oligoribonucleotides and improves predictions of RNA folding. Proc Nat/ Acad Sci USA 1994, 91:9218-9222. 2.
Chastain M, Tinoco I: Nucleoside triples from the group I intron. Biochemistry 1993, 32:14220-14228.
3.
Reid RiD, Zhang F, Benson S, Anderson D: Probing the structure of bacteriophage ~29 prohead RNA with specific mutations. J Biol Chem 1994, 269:18656-18661.
4. Lorsch JR, Szostak JW: In vitro selection of RNA aptamers ,, specific for cyanocohalamin. Biochemistry 1994, 33:973-982. The authors describe the in vitro selection of an unusual pseudoknot that binds an enzyme cofactor believed to be of ancient origin. This example is of particular interest because lithium is involved in the folding of the selected RNA molecule. 5.
Torrent C, Bordet 1", Darlix J-L: Analytical study of rat retrotransposon VI.30 RNA dimerizatlon and packaging in murlne leukemia virus. J Mol Biol 1994, 240:434-444.
Battiste JL, Tan R, Frankel AD, Williamson JR: Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base pairs. Biochemistry 1994, 33:2741-2747. See annotation [7••].
6. ••
7. ••
Peterson RD, Barrel DP, Szostak JW, Horvath SJ, Feigon ]: IH NMR studies of the high-affinity Rev binding site of the Rev responsive element of HIV-1 mRNA: base pairing in the core binding element. Biochemistry 1994, 33:5357-5366. This article and [6 •• ] are outstanding papers that describe the peptide-binding site called the Rev responsive element within an RNA. Upon binding of peptide, an asymmetric internal loop within the RNA undergoes a large conformational change, forming a helical structure with exposed functional groups in the major groove. Gautheret D, Konings D, Gutell RR: A major family of motifs involving G-A mismatches in ribosomal RNA. J Mol Biol 1994, 242:1-8. This paper succeeds in defining G-A pairs and tandem pairs as a general motif important for RNA folding and molecular recognition. 8. •
9. Butcher SE, Burke JM: Structure mapping of the hairpin • ribozyme. J ~viol Biol 1994, 244:52-63. See annotation [10•]. Butcher SE, Burke JM: A pholo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme. Biochemistry 1994, 33:992-999. A ubiquitous family of asymmetric loops is now believed to contribute to the structure and, in some cases, the catalytic properties of RNA. One example of this motif is loop B, found in the hairpin ribozyme. This paper and [9 • ] provide structural insight into the morphology of this important new RNA folding domain. 10. •
Tang RS, Draper DE: Bend and helical twist associated with a symmetric internal loop from 5S ribosomal RNA, Biochemistry 1994, 33:10089-10093. Although symmetric internal loops are generally thought to be straight, this paper shows that certain symmetric loop motifs can adopt rigid, bent conformations. 11. •
12. Strobel SA, Cech TR: Translocalion of an RNA duplex on a • ribozyme. Nature Struct Biol 1994, 1:13-17. The 2'-hydroxyl groups along an RNA helix can form a recognition element, docking as a group into binding pockets within folded RNA molecules. This paper extends earlier analyses of this motif, showing that the recognition helix can translocate, docking 2'-hydroxyl groups into alternative binding registers. 13. ••
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest *• of outstanding interest 1.
Walter AE, Turner DH, Kim J, I_yttle MH, MLiller P, Mathews DH, Zucker M: Coaxial stacking of helices enhances binding of
Pyle AM, Moran S, Strobel SA, Chapmen 1", Turner DH, Cech TR: Replacement of the conserved G-U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity and fidelity. Biochemistry 1994, 33:13856-13863. See annotation [14••].
Knitt DS, Narlikar GJ, Herschlag D: Dissection of the role of the conserved G-U pair in group I RNA self-splicing. Biochemistry 1994, 33:13864-13879. This article and [13 •'] describe the role that G-U pairs may play in the docking of recognition helices within large RNA molecules. They show that the formation of important tertiary interactions and the selection of 14. •*
R N A folding Pyle and Green a ribozyme cleavage site are linked to recognition of a conserved G-U wobble pair. 15. •
Walter AE, Turner DH: Sequence dependence of stabilily for coaxial stacking of RNA helixes with Watson-Crick base paired interfaces. Biochemistry 1994, 33:12715-12719. Coaxial stacking is an essential architectural element in folded RNA molecules, and Walter and Turner succeed in describing the sequence dependence of this process.
27.
Komatsu Y, Koizumi M, Nakamura H, Ohtsuka E: Loop-size variation to probe a bent structure of a hairpin ribozyme. J Am Chem Soc 1994, 116:3692-3696.
28.
Tanner NK, Schaff S, Thill G, Petit-Koskas E, Crain-Denoyelle A-M, Westhof E: A three-dimensional model of hepatitis delta virus rlbozyme based on biochemical and mutational analyses. Curt Biol 1994, 4:488-498.
29. Murphy FL, Wang Y-H, Griffith JD, Cech TR: Coaxially stacked RNA helices in the catalytic center of the Tetrahymena ribozyme. Science 1994, 265:1709-1712. The structural effect of an essential coaxial stack can be directly visualized in this electron microscopic study of the Tetrahymena ribozyme.
Kumar PKR, Taira K, Nishikawa S: Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis. Biochemistry 1994, 33:583-592.
30.
Gottlieb PA, Prasad Y, Smith JB, Williams AP, Dinter-Gottlieb G: Evidence that alternate foldings of the hepatitis delta RNA confer varying rates of self-cleavage. Biochemistry 1994, 33:2802-2808.
17. •
31.
Steinberg S, Gautheret D, Cedergren R: Fitfing the structurally diverse animal mitochondrial IRNA set to common three-dimensional constraints. J ?viol Biol 1994, 236:982-989.
18. •"
32. •
19. +"
33. Pley HW, Flaherty KM, McKay DB: Three-dimensional structure "" of a hammerhead ribozyme. Nature 1994, 372:68-74. The first crystal structure of a complex RNA molecule other than tRNA. This classic paper is also significant because there is considerable enzymological information on the hammerhead ribozyme, which is useful in interpreting the structure.
20. *
34. Amid KMA, Hagerman PJ: Global conformation of a self-cleav• ing hammerhead RNA. Biochemistry 1994, 33:13172-13177. The authors use transient electric birefringence, a promising structural technique, to analyze the overall structure of the hammerhead ribozyme. Useful for comparison with the crystal and fluorescence structures of the ribozyme.
16. "*
Murphy FL, Cech TR: GAAA tetraloop and conserved bulge stabilize tertiary structure of a group I intron domain. I Mol Biol 1994, 236:49-63. See annotation [18"']. Jaeger L, Michel F, Westhof E: Involvement of a GNRA tetraloop in long-range RNA tertiary interadions. J Mol Biol 1994, 236:1271-1276. This paper and [17•] define the helix-docked tetraloop motif, which is a long-range RNA tertiary interaction that contributes to the three-dimensional organization of many different RNA molecules. This general structure is of fundamental importance in RNA tertiary folding. Pley HM, Flaherty KM, McKay DB: Model for an RNA tertiary interaction from the structure of an inlermolecular complex between a GAAA tetraloop and an RNA helix. Nature 1994, 372:111-113. A high-resolution analysis of the helix-docked tetraloop motif, provided by the crystal structure of the hammerhead ribozyme. Chang K-Y, Tinoco I: Characterization of a "kissing" hairpin complex derived from the HIV genome. Proc Nat/ Acad Sci USA 1994, 91:8705-88709. This paper shows that the loop nucleotides of two hairpin RNAs can associate and drive the formation of a two-hairpin complex. 21. •
Pan T: Higher-order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry 1995, 34:902-909. See annotation [22*].
22. •
Pyle AM, Green JB: Building a kinetic framework for group II intron ribozyme activity: quantitation of interdomain binding and reaction rate. Biochemistry 1994, 33:2716-2725. This paper and [21 +} describe the assembly of large ribozymes from separate RNA domains, which form active complexes that can be studied using enzymological approaches. In this way, information about the tertiary architecture and interactions important to RNase P and group II intron folding was determined.
Malhotra A, Tan RKZ, Harvey SC: Modeling large RNAs and ribonucleoprotein parlicles using molecular mechanics techniques. Biophys J 1994, 66:1777-1795. An excellent discussion of current approaches and considerations important in the computer modeling of complex RNA structures.
35. "
Tuschl T, Gohlke C, Jovin TM, Westhof E, Eckstein F: A threedimensional model for the hammerhead ribozyme based on fluorescence measurements. Science 1994, 266:785-789. This meticulous application of fluorescence resonance energy transfer (FRET), combined with molecular modeling procedures, demonstrates the power of FRET for solution structural studies of RNA. The study provides an interesting comparison with the crystal structure of the ribozyme. 36. •
Woisard A, Fourrey J-L, Favre A: Multiple folded conformations of a hammerhead ribozyme domain under cleavage conditions. J Mol Biol 1994, 239:366-370. In light of various structural models for the hammerhead ribozyme [33"~,34•,35"] this work gives a physical description of conformational heterogeneity in the molecule.
23. Laing LG, Gluick TC, Draper DE: Stabilization of RNA structure " by Mg ions. J Mol Biol 1994, 237:577-587. A remarkable analysis of the three different roles that metal ions can play in the stabilization of RNA tertiary structure.
37. •
24. •
38. •
Yue D, Kintanar A, Horowitz J: Nucleoside modifications stabilize Mg 2+ binding in E. coli tRNAval: an imino proton NMR investigation. Biochemistry 1994, 33:8905-8911. Post-transcriptional base modifications were not previously thought to play a large role in the folding of tRNA; however, this study shows that modifications can be involved in the binding of stabilizing metal ions. 25.
26. *"
Arnez JG, Steitz TA: Crystal structure of unmodified IRNA GIn complexed with glulaminyl-tRNA synthelase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry 1994, 33:7560-7567.
Westhof E, Ahman S: Three-dimensional working model of Mt RNA, the catalytic RNA subunit of ribonuclease P from E. coli. Proc Nat/ Acad 5ci USA 1994, 91:5133-5137. See annotation [38"].
Harris ME, Nolan JM, Malhotra A, Brown JW, Harvey SC, Pace NR: Use of photoaffinity crosslinking and molecular modeling to analyze the global archileclure of ribonuclease P RNA. EMBO J 1994, 13:3953-3963. This paper and [37*] describe three-dimensional models of RNase P generated by the use of biochemical and phylogenetic techniques combined with computational approaches to RNA tertiary structure modeling. 39.
Mattson JG, Svard SG, Kirsebom LA: Charaderization of the Borrelia burgdorferl RNase P RNA gene reveals a novel tertiary interaction. J Mol Biol 1994, 241:1-6.
40.
Franzen JS, Zhang M, Chay TR, Peebles CL: Thermal activation of a group II inlron rihozyme reveals multiple conformational states. Biochemistry 1994, 33:11315-11326.
41. •"
Wang YH, Murphy FM, Cech TR, Griffith JD: Visualization of a tertiary structural domain of the Tetrahymena group I intron by electron microscopy. J Mol Biol 1994, 236:64-71.
Kowalak JA, Dalluge JJ, McCIoskey JA, Stetter KO: The
role of posttranscriplional modification in stabilization of transfer RNA from hyperthermoflbiles. Biochemistry 1994, 33:7869-7876. By exploring the role of base modifications in the folding of tRNAs from thermophitic organisms, the authors make important observations about the basis for thermodynamic stabilization and the folding pathways of RNA molecules.
309
31 0
Nucleic acids Mutagenesis and electron microscopy are combined, revealing a dramatic kinked conformation essential for activity of a group I intron structural domain.
42.
Burd CG, Dreyfuss G: Conserved structures and diversity of functions of RNA-binding proteins. Science 1994, 265:615-621. A useful reference for understanding the emerging field of RNA-protein interactions. ,
43. ""
Mohr G, Caprara MG, Guo Q, Lambowitz AM: A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme. Science 1994, 370:147-150. This study establishes an important new concept by demonstrating that a protein, provided in trans, can substitute for an essential RNA substructure in the Tetrahymena ribozyme. In so doing, the study underscores parallels in molecular recognition by RNA and proteins. 44. ""
Hall KB: Interaction of RNA hairpins with the human UIA N-terminal RNA binding domain. Biochemistry 1994, 33:10076-10088. See annotation [45ee]. 45. ""
48. ""
Herschlag D, Khosla M, Tsuchihashi Z, Karpel RL: An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis. EMBO J 1994, 13:2913-2924. Just as proteins can act as chaperones for protein folding, so can they also chaperone RNA folding, by selectively stabilizing particular RNA conformations. The concept is illustrated in this study of the effects of nucleocapsid protein on the kinetics of folding of hammerhead ribozymes. 49. •
Laggerbauer B, Murphy FL, Cech TR: Two major tertiary folding transitions of the Tetrahymenacatalytic RNA. EMBO J 1994, 13:2669-2676. See annotation [50ee]. 50. Zarrinkar PP, Williamson JR: Kinetic intermediates in RNA ee folding. Science 1994, 265:918-924. The folding pathway of the Tetrahymenaribozyme is described in this paper and [49"]. This paper is of considerable interest because it shows that the folding of a single RNA structural domain limits the rate of folding for the entire ribozyme. 51. ""
Laing LG, Draper DE: Thermodynamics of RNA folding in a conserved ribosomal RNA domain. J Mol Biol 1994, 237:560-576. See annotation [52e"].
Oubridge C, Ito N, Evans PR, Teo C-H, Nagai K: Crystal structure at 1.92A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature 1994, 372:432--438. Together, crystallographic studies (this paper) and NMR studies [44 e'] of RNA binding to the U1A protein provide a clear view of an RNA-protein interface, which is one of the first (other than the synthetase-tRNA interaction) to have been characterized.
52. Gluick TC, Draper DE: Thermodynamics of folding a pseudo•e knotted mRNA fragment. J Mol Biol 1994, 241:246-262. Together, this paper and [51"'] show that different global folding pathways will be adopted by different RNA molecules. They both show that the folding of secondary and tertiary structural elements can be interdependent.
46.
53.
47. e.
Nureki O, Niimi T, Muramatsu I, Kanno H, Kohno T, Florentz C: Molecular recognition of the identity-determinant set of isoleucine transfer RNA from E. coil ] Mol Biol 1994, 236:71 0-724.
Tan R, Frankel AD: Costahilization of peptide and RNA structure in an HIV Rev peplide-RRE complex. Biochemistry 1994, 33:14579-14585. The authors provide evidence that RNAs and proteins can co-fold, a concept that unites the respective problems of RNA and protein folding.
Cech TR, Damberger SH, Gutell RR: Representation of the secondary and tertiary structure of group I inlrons. Nature Struct Biol 1994, 1:273-280.
AM Pyle and JB Green, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Introduction In spite of its deceptively simple primary structure, R N A carries out a broad spectrum of biological functions beyond its informational role in gene expression. We now know that catalytic R N A molecules (ribozymes) are capable of catalyzing numerous chemical transformations. A large number of R N A structural motifs are now being recognized to be important in regulating diverse biochemical processes, including the translation of particular genes and the expression of viral genomes. Consequently, it is important for us to expand our understanding of R N A folding. In this review, we detail work in the past year on the building blocks for folding, and follow it with a section on how these units are assembled. The effects of metals and base modifications on P,.NA folding are assessed. New information on the folds of particular RNAs is provided, together with a section on the important new topic ofribonucleoprotein folding. All of this is concluded with a review of new information on the pathways and order of events involved in R N A folding.
Architectural elements of RNA folding: defining the building blocks The arrangement of R.NA structural elements in three-dimensional space is achieved by a process called R N A tertiary folding, The most fundamental structural elements as far as folding of R N A is concerned are the helical regions of secondary structure, which have stabilities that can be calculated with reasonable accuracy [1]. Considerable effort is being expended on defining additional units of P.NA folding. These motif~ are often stable enough to have biological functions of their own, but they may also function within the context of larger RNAs. They include particular kinds of pseudoknots,
loops that cap helices, loops within helices, regions of R N A mispairing, nucleoside triple interactions [2], quadruplexes and U-turns. Pseudoknots are interlocked regions of coaxially stacked helices that are commonly involved in R N A binding and folding. A pseudoknot structure proposed for regions of bacteriophage (I)29 prohead R N A is involved in DNA packaging [3]. Pseudoknotting is a motif with a high capacity for molecular recognition, as shown by the large number of pseudoknot structures derived from in vitro selection schemes. For example, an R N A with a high affinity for cyanocobalamin is proposed to have a pseudoknot structure [4°]. This interaction is particularly dependent on lithium ions, which are so small and unshielded that they can allow tight packing of phosphate residues within the folded RNA. An unusual motif in rat retrotransposon VL30 I:(NA was also proposed to fold specifically in the presence of Li +, so the effect of this ion may be general [5]. The P,.ev responsive element (tkR~) of HIV has an open internal loop of R N A that undergoes a conformational change upon binding of the Rev protein. In the complex, the Ioop region forms an underwound helix made up of purine-purine pairs that dramatically widen the major groove of the R N A [6"',7°']. This wide major groove of unusual accessibility may be a general feature imposed by tracts of G--G and G-A pairs imbedded within a helix. Although base functional groups in the major groove are not exposed in normal RNA helices, the R R E motif may be one strategy for increasing the accessibility of the numerous functional groups located m the major groove of R N A helices. Gautheret et al. [8"] observed that phylogenetically conserved G-A mismatches occur singly or in patterns of two tandem G - A mismatches. They proposed that by exposing purine functional groups to the solvent, these G--A motifs ,nay facilitate R N A tertiary interactions or protein binding [8°].
Abbreviations FRET~fluorescence resonance energy transfer, HDV--hepatitis delta virus; RRE--Rev responsiveelement,
© Current Biology Ltd ISSN 0959-440X
303
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Nucleicacids Another group of internal loops that constitute a distinct and ubiquitous R N A folding motifs include structures variously termed Loop B (hairpin ribozyme), Loop E (eukaryotic 5S RNA) and the sarcin/ricin loop (16S RNA). The sequence of this asymmetrical loop motif is highly conserved [9"]. UV irradiation of the hairpin ribozyme results in a highly efficient cross-link across the loop B region [10"]. This result, together with those from modification interference studies, shows that the motif has a distinctive tertiary architecture [9°]. Electrophoretic phasing experiments on the symmetric Escherichia coli 5S R N A Loop E demonstrated the presence of a rigid bend within an overall helical structure [11"]. Despite the bending and the overwinding of the helix by 27 °, none of the bases were pushed out of the helix and the structure remained stacked.
Assembly of motifs into tertiary structures It is often necessary for individual blocks of R N A secondary structure to condense into tertiary structure. The folding of structural elements takes place through the formation of long-range tertiary interactions, which are now being defined in terms of discrete molecular patterns of chemical bonding. The backbone substituents that line the outside of R N A helices are important points of tertiary contact. In particular, ribose U-hydroxyls can bond directly to a variety of substitutents, setting a helical element into place within large R N A structures. This type of bonding arrangement appears to be particularly significant when the interactions mediated by T-hydroxyls occur in groups, as shown in a recent examination of tertiary interactions between the substrate recognition helix and the core of the Tetrahymena ribozyme ([12"]; see also Wahl and Sundaralingam, pp 282-295 and Tuschl et al., pp 296-302, this issue). Because all ribose residues have a U-hydroxyl group, additional elements may be required to control the specificity of U-hydroxyl interactions within the tertiary structure. In the case of the Tetrahymena ribozyme, a conserved G - U wobble pair is situated at the ribozyme cleavage site, immediately adjacent to an array of important U-hydroxyls. When this G--U pair is replaced with a G--C pair, the 2'-hydroxyl groups no longer dock into place and the enzymatic behavior of the ribozyme is significantly disrupted [13°%14°°]. The coaxial stacking of helices can fundamentally direct the overall fold of an RNA. The sequence dependence of coaxial stacking was recently analyzed and observed to follow the same nearest-neighbor rules that govern the formation of contiguous helices [15°]. For example, if there are three helices arranged in space with similar intervening constraints, one would predict that the two that come together in a coaxial stack may have terminal base pairs with the highest favorable fi:ee energy for
stacking, such as two G - C pairs, rather than a G - C pair and an A - U pair. The P4-P6 coaxial stacking interaction has a dominant effect on the overall fold and dimensions of the Tetrahymena ribozyme [16"'] (Fig. 1). A striking long-range tertiary interaction, involving specific binding of G N R A tetraloops (where N is any nucleotide and R is any purine) to G--C pairs within helical regions, was recently identified in a wide variety of different R N A molecules. Modification interference analysis on the Tetrahymena ribozyme and deletion mutants of it suggested that a tertiary interaction takes place between the GAAA tetraloop of P5abc (Fig. 1) and a C - G pair in region P6a [17"]. In the td group I intron, mutational and kinetic analysis showed that bases 3 and 4 of a conserved GUGA tetraloop specifically recognize nucleotide functional groups in the minor groove of two stacked A - U and G - C base pairs [18"°]. The tetraloop-helix docking motif was visualized at high resolution during crystallographic analysis of the hammerhead ribozyme (Fig. 2). Although the ribozyme itself does not contain it, the motif was formed as a consequence of intermolecular interactions between two ribozyme molecules. In this case, the principal interactions occured between GAAA tetraloop bases and two stacked G--C pairs on another molecule [19"']. Certain types of hairpin loops can bind to each other, as shown by N M R studies of a "kissing hairpin" [20°]. Circular permutation analysis of RNase P and domains of the Tetrahymena ribozyme reveals that the position of 5'- and 3'-ends in a correctly folded and functionally active R N A can be somewhat arbitrary [16"',21"]. This implies that location of the ends is not necessarily an element of importance in tertiary folding.
Trans domain analysis provides a way to quantitate the effects of tertiary interactions and folding of large R N A molecules. Once the independent folding domains of an R N A are determined, they may be transcribed as separate pieces that associate solely through tertiary interactions. If the R N A has catalytic activity, kinetics and binding studies can be used to evaluate the interaction energies and mechanistic role of specific folding domains. The mechanistic role of catalytic domain 5 (D5) of the group II intron was evaluated in this way. Despite the apparent lack of canonical base-pairing interactions between D5 and other components of the intron, D5 was observed to bind strongly to them, with a Kd of approximately 300 nM [22"]. A kinetic framework for D5 reactivity was also established that facilitates dissection ofsubstituent effects on either tertiary interaction energy or chemistry. Similarly, separation of RNase P into two domains facilitated investigation of metal binding and folding energetics in that molecule [21°]. The P4-P6 domain of the Tetrahymena ribozyme is an independently folding domain that can also be provided in trans to remaining portions of the molecule [17"]. Its contribution to ribozyme folding was monitored by modification interference and electron microscopy.
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The role of metal ions and base modifications in folding Metal ions play a critical role in folding and catalysis by R N A , although their effects have remained difficult to define because metals contribute to R N A stabilization in several ways. These distinct functions have been recently clarified through thermal denaturation experiments on different types of R N A molecules [23°']. Laing et al. [23 °°] show that if metal ions are binding an R N A non-specifically, thus playing a role in charge screening and duplex formation, a plot of the reciprocal of the melting temperature of the R N A (Tm-I) against [Mg2+] will be sigmoidal. If metal ions bind an R N A site-specifically, this plot will be linear. R N A molecules with a high degree of tertiary structure show this linear dependence. In one R N A , linear behavior was specific for Mg 2+, whereas in another, Co(NH3)63+ could substitute for Mg2+. This shows that there are
Fig. 1. Secondary structural representation of the Tetrahymena intron (upper case letters) and flanking exon sequences (lower case letters). The P1 duplex Iocatecl in the middle of this schematic contains the 5"-splice site or cleavage site, marked by an arrow at the conserved G - U wobble pair. The second arrow (near Pg.0) indicates the position of the 3'-splice site. Flanking the PI duplex are folded core elements, including the P4-P6 domain to the left and the P3-P7 domain to the right. The P5abc domain is shown to the left of P4-P6, with which it interacts. Thick lines represent continuity of the strands, and no nucleotides have been omitted. Arrowheads indicate the 5'--)3' polarity of the strands. Base pairings are represented by dark dashes. The light dashes extending from some of the bases indicate positions of known tertiary contact. Reproduced from [53] with permission.
at least three general roles for metals in R N A folding: non-specific binding; specific Mg2+ coordination; and binding to high-affinity sites through charge density or outer-sphere coordination rather than through the formation of direct metal contacts. In vivo, most functional R N A s are modified posttranscriptionally with a variety of functional groups. These are now known to have important effects on R N A folding. N M R studies comparing unmodified with modified tRNA^la show that base modifications strengthen metal-binding sites [24°]. A recent crystal structure of unmodified tRNAPhe shows that it is missing a water molecule near the nucleotide normally occupied by a W-base [25]. This suggests that modifications may influence the organization of water and thereby influence macromolecular stability.
An interesting report about modifications that reflects generally on the problem o f R N A folding involves
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Fig. 2. The long-range tertiary interaction between a GAAA tetraloop and a tandem G~C pair determined by crystallography. (a) A schematic of major stem interactions between two molecules of the hammerhead ribozyme in the unit cell. (b) A high-resolution illustration of the interactions of loop 4 adenosine (AL4 , on molecule 1 in [a]) with the GT4-CT4 base pair (on molecule 2). Note the abundance of base-backbone interactions and the additional intraloop contacts within molecule 1, between G U and AL4. Reproduced from [33 °°] with permission.
tRNA from hyperthermophilic bacteria [26°°]. As the temperature o f bacterial culture medium was raised, t R N A became modified more extensively. Three modifications known to promote greater helical rigidity (and in some cases better stacking) became especially prevalent. The modifications helped stabilize secondary structure. Interestingly, once tertiary structure was formed in these variously modified RNAs, they all had similar melting temperatures. This suggests that the modifications may be important along a folding pathway, rather than in maintenance o f folded structure.
Folding of specific RNAs A number o f recent papers have provided important information on the conformation and structure o f specific R N A molecules, particularly the smaller ribozymes and tRNA. Linkers were inserted between domains [27], and chemical modification studies were conducted [9°] in order to probe the overall folding of
the hairpin ribozyme and the dependence o f the process on Mg 2+. High-temperature analysis of the structure of genomic hepatitis delta virus (HDV) ribozyme resulted in greater sensitivity of mutational analysis and facilitated development of more refined structural models [28]. Chemical probing [29] and deletional studies on forms o f HDV ribozyme [30] revealed additional information about HDV folding and stability. Molecular modeling studies provided new proposals for the folding o f unusual mitochondrial tRNAs [31] and for the construction of three-dimensional models of large RNAs [32°]. Some of the most detailed information available on R N A folding comes from the first crystal structure of a ribozyme, reported in 1994. Before determination of the structure o f the hammerhead ribozyme [33°°], our only detailed view of folded R N A came from crystal structures o f tRNA. In the hammerhead structure, several core nucleotides form a set of G - A pairs that stack onto stem II of the ribozyme (Fig. 3). This structure is coaxially stacked with stem III. The ribozyme then forms a roughly Y-shaped structure as stem I curves alongside stem II and forms a bed of core nucleotides in the junction between the stern I and stem II helices. The unpaired core nucleotides form a defined R N A motif known as the "U-turn", which is identical in conformation to the uridine turn in the anticodon of t R N A Phe. The relative orientation o f the hammerhead stems was also examined using transient electric birefringence measurements [34°]. Fluorescence resonance energy transfer (FRET) measurements combined with molecular modeling provided an additional detailed structure of the hammerhead ribozyme [35°°]. Although the helical orientations and global conformation determined by F R E T are similar to those in the crystal structure, the conformation of core nucleotides differs. There are salient differences between the molecules used in the two studies that may have affected their overall structures. A recent cross-linking study provided evidence for multiple tertiary conformations of the hammerhead ribozyme [36°], and this structural variability may explain differences between recent models of the ribozyme. The conformation and folding of larger RNAs was also the subject o f many investigations in the past year. Results from chemical protection studies, phylogenetic analyses, mutagenesis and kinetics were combined to produce a working model of RNase P tertiary structure [37°]. Results from photoaffinity cross-linking o f circularly permuted RNase P was also used to generate a second structural model [38°]. Although the models appear superficially different, they share many common features and differ only in the relative orientations of certain subdomains of the molecule. Phylogeny and mutagenesis studies revealed a long-range tertiary interaction across the large loop o f R N a s e P [39], a feature that may help to further constrain the structure. Conformational variability of domains within the group II intron was explored through a combination o f kinetic and thermal denaturation experiments [40]. The folding
RNA folding Pyle and Green 307
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of group I intron domains was visualized directly using electron microscopy of the Tetrahymena ribozyme. This study provided direct proof o f the presence of a sharp kink believed to exist between two rigid portions of the P5 domain [41°'].
Ribonucleoprotein folding Although the intrinsic properties of R N A molecules enable them to act as catalysts and units of molecular recognition, most of them function together with proteins in vivo. Given the intimate cooperation that exists between many R N A s and proteins, it may be increasingly important to consider a more unified concept in macromolecular organization: ribonucleoprotein folding. As the term implies, certain R N A s and proteins may fold together, making it important to unite concepts in R N A and protein folding. Specific classes of ribonucleoprotein folding have been demonstrated in recent examples from the literature and a recent review describes known motifs for RNA-protein interaction [42% The Tetrahymena ribozyme contains a large, independently folding domain known as P5abc, which can participate in the folding and activation of the ribozyme even when added in trans (Fig. 1). It was shown that this same function can also be provided by a protein known as Cyt-18, a tyrosyl t R N A synthetase [43"°]. The protein specifically binds and activates the
ribozyme only if it is missing the P5abc domain. This work conclusively demonstrates that certain RNAs and proteins can perform the same roles in stabilizing the active tertiary fold of an R N A molecule. The interaction of U1A protein with an R N A hairpin provides an example of an R N A molecule that folds only in the presence of a protein. In this example of induced fit, N M R and crystallographic studies reveal that a loop sequence in the R N A undergoes a massive conformational change [44°'], binding to the surface of an exposed J-sheet through many hydrogen-bonding contacts and base-stacking with a tyrosyl side chain [44°',45°']. In this example, the protein structure changes little upon R N A binding. Recent studies of t R N A lle suggest that certain R N A tertiary interactions are actually broken and new ones are formed upon synthetase binding [46]. A very different story is revealed in biochemical analyses of R e v - R R E binding. This RNA-protein interaction may best be described as a 'co-folding', in which association results in radical conformational changes in both R N A and protein [47°°]. Detailed kinetic analyses reveal that nucleocapsid protein can function as a chaperone for R N A folding [48°°]. The protein has effects on duplex annealing, stimulating product dissociation rate from the hammerhead ribozyme and thereby increasing the multiple-turnover rate of reaction (kcat). Nucleocapsid protein acts as a folding chaperone by eliminating a 'misfolded trap', or less-active conformation, that can be adopted by the ribozyme-substrate complex. This demonstrates that even non-specific RNA-binding proteins can function as R N A chaperones.
Pathways for RNA folding Now that motifs for local and tertiary folding of R N A have been described, we can attempt to explain how an R N A molecule gets to the folded state. R N A folding is believed to be distinct from protein folding in that R N A secondary structural elements are very stable and capable of forming independently of tertiary structure. This has led to the concept that R N A secondary structure forms rapidly and precedes the packaging of R N A into tertiary structure. Recent work shows that this idea needs to be extended in new directions and that different RNAs will not necessarily follow this canonical pathway. An important modification of RNA-folding theory is the need to recognize a hierarchy of individual RNA-folding domains. For example, in the Tetrahymena ribozyme, folding of the catalytic core is dependent upon formation of the independent P4-P6 domain [49°]. The folding pathway for the ribozyme was dissected using time-resolved oligonucleotide probe hybridization [50°°]. This study showed that the P4--P6 domain forms first, but that the rate-limiting step in overall folding is
308
Nucleicacids assembly of the P3-P7 domain. Once P3--P7 is folded, global tertiary organization rapidly follows. Thus, the rate-limiting step in this folding pathway is the formation of a domain, rather than tertiary organization of the final state. It is not yet clear whether formation of secondary or tertiary structures actually limits the rate of P3-P7 folding. In a model for the unfolding of a domain in the R,NA of the large ribosomal subunit [51•'], the pathway is dissected into five sequential transitions. The first transition is the unfolding of the magnesium-dependent tertiary structure, which occurs before the unfolding of secondary structure. The other transitions represent the coupled unfolding of four different helical units. Although this example appears to follow the standard paradigm for R N A folding, it is interesting that the secondary structural transitions are linked to formation of a specific pairing interaction in the junction loop between helices. The notion that I~NA tertiary structure always folds last is challenged by studies on the unfolding pathway of a complex pseudoknot [52°°]. In that case, the tertiary structure was not easily separated from the secondary structure and it is proposed that tertiary interactions may be required for the formation of a secondary structure element that was unstable on its own.
Conclusion The motifs and thermodynamic basis for R N A folding received considerable attention during the past year. This is partly because of an abundance of new structural information. The first crystal structure of a large R N A molecule other than tlLNA was reported [33°°]. This structural investigation of the hammerhead ribozyme provided a new window into overall R N A architecture and also revealed high-resolution information on longrange tertiary interactions. Numerous NMI:( studies revealed dramatic conformational changes in R N A and, together with biochemical and crystallographic studies, set the stage for new thinking on ribonucleoprotein folding. Throughout the year, spectroscopic and biochemical studies continued to elucidate the motifs that are fundamental to R N A folding, showing how they coalesce and interact to shape the tertiary structure of an R N A molecule.
oligoribonucleotides and improves predictions of RNA folding. Proc Nat/ Acad Sci USA 1994, 91:9218-9222. 2.
Chastain M, Tinoco I: Nucleoside triples from the group I intron. Biochemistry 1993, 32:14220-14228.
3.
Reid RiD, Zhang F, Benson S, Anderson D: Probing the structure of bacteriophage ~29 prohead RNA with specific mutations. J Biol Chem 1994, 269:18656-18661.
4. Lorsch JR, Szostak JW: In vitro selection of RNA aptamers ,, specific for cyanocohalamin. Biochemistry 1994, 33:973-982. The authors describe the in vitro selection of an unusual pseudoknot that binds an enzyme cofactor believed to be of ancient origin. This example is of particular interest because lithium is involved in the folding of the selected RNA molecule. 5.
Torrent C, Bordet 1", Darlix J-L: Analytical study of rat retrotransposon VI.30 RNA dimerizatlon and packaging in murlne leukemia virus. J Mol Biol 1994, 240:434-444.
Battiste JL, Tan R, Frankel AD, Williamson JR: Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base pairs. Biochemistry 1994, 33:2741-2747. See annotation [7••].
6. ••
7. ••
Peterson RD, Barrel DP, Szostak JW, Horvath SJ, Feigon ]: IH NMR studies of the high-affinity Rev binding site of the Rev responsive element of HIV-1 mRNA: base pairing in the core binding element. Biochemistry 1994, 33:5357-5366. This article and [6 •• ] are outstanding papers that describe the peptide-binding site called the Rev responsive element within an RNA. Upon binding of peptide, an asymmetric internal loop within the RNA undergoes a large conformational change, forming a helical structure with exposed functional groups in the major groove. Gautheret D, Konings D, Gutell RR: A major family of motifs involving G-A mismatches in ribosomal RNA. J Mol Biol 1994, 242:1-8. This paper succeeds in defining G-A pairs and tandem pairs as a general motif important for RNA folding and molecular recognition. 8. •
9. Butcher SE, Burke JM: Structure mapping of the hairpin • ribozyme. J ~viol Biol 1994, 244:52-63. See annotation [10•]. Butcher SE, Burke JM: A pholo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme. Biochemistry 1994, 33:992-999. A ubiquitous family of asymmetric loops is now believed to contribute to the structure and, in some cases, the catalytic properties of RNA. One example of this motif is loop B, found in the hairpin ribozyme. This paper and [9 • ] provide structural insight into the morphology of this important new RNA folding domain. 10. •
Tang RS, Draper DE: Bend and helical twist associated with a symmetric internal loop from 5S ribosomal RNA, Biochemistry 1994, 33:10089-10093. Although symmetric internal loops are generally thought to be straight, this paper shows that certain symmetric loop motifs can adopt rigid, bent conformations. 11. •
12. Strobel SA, Cech TR: Translocalion of an RNA duplex on a • ribozyme. Nature Struct Biol 1994, 1:13-17. The 2'-hydroxyl groups along an RNA helix can form a recognition element, docking as a group into binding pockets within folded RNA molecules. This paper extends earlier analyses of this motif, showing that the recognition helix can translocate, docking 2'-hydroxyl groups into alternative binding registers. 13. ••
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest *• of outstanding interest 1.
Walter AE, Turner DH, Kim J, I_yttle MH, MLiller P, Mathews DH, Zucker M: Coaxial stacking of helices enhances binding of
Pyle AM, Moran S, Strobel SA, Chapmen 1", Turner DH, Cech TR: Replacement of the conserved G-U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity and fidelity. Biochemistry 1994, 33:13856-13863. See annotation [14••].
Knitt DS, Narlikar GJ, Herschlag D: Dissection of the role of the conserved G-U pair in group I RNA self-splicing. Biochemistry 1994, 33:13864-13879. This article and [13 •'] describe the role that G-U pairs may play in the docking of recognition helices within large RNA molecules. They show that the formation of important tertiary interactions and the selection of 14. •*
R N A folding Pyle and Green a ribozyme cleavage site are linked to recognition of a conserved G-U wobble pair. 15. •
Walter AE, Turner DH: Sequence dependence of stabilily for coaxial stacking of RNA helixes with Watson-Crick base paired interfaces. Biochemistry 1994, 33:12715-12719. Coaxial stacking is an essential architectural element in folded RNA molecules, and Walter and Turner succeed in describing the sequence dependence of this process.
27.
Komatsu Y, Koizumi M, Nakamura H, Ohtsuka E: Loop-size variation to probe a bent structure of a hairpin ribozyme. J Am Chem Soc 1994, 116:3692-3696.
28.
Tanner NK, Schaff S, Thill G, Petit-Koskas E, Crain-Denoyelle A-M, Westhof E: A three-dimensional model of hepatitis delta virus rlbozyme based on biochemical and mutational analyses. Curt Biol 1994, 4:488-498.
29. Murphy FL, Wang Y-H, Griffith JD, Cech TR: Coaxially stacked RNA helices in the catalytic center of the Tetrahymena ribozyme. Science 1994, 265:1709-1712. The structural effect of an essential coaxial stack can be directly visualized in this electron microscopic study of the Tetrahymena ribozyme.
Kumar PKR, Taira K, Nishikawa S: Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis. Biochemistry 1994, 33:583-592.
30.
Gottlieb PA, Prasad Y, Smith JB, Williams AP, Dinter-Gottlieb G: Evidence that alternate foldings of the hepatitis delta RNA confer varying rates of self-cleavage. Biochemistry 1994, 33:2802-2808.
17. •
31.
Steinberg S, Gautheret D, Cedergren R: Fitfing the structurally diverse animal mitochondrial IRNA set to common three-dimensional constraints. J ?viol Biol 1994, 236:982-989.
18. •"
32. •
19. +"
33. Pley HW, Flaherty KM, McKay DB: Three-dimensional structure "" of a hammerhead ribozyme. Nature 1994, 372:68-74. The first crystal structure of a complex RNA molecule other than tRNA. This classic paper is also significant because there is considerable enzymological information on the hammerhead ribozyme, which is useful in interpreting the structure.
20. *
34. Amid KMA, Hagerman PJ: Global conformation of a self-cleav• ing hammerhead RNA. Biochemistry 1994, 33:13172-13177. The authors use transient electric birefringence, a promising structural technique, to analyze the overall structure of the hammerhead ribozyme. Useful for comparison with the crystal and fluorescence structures of the ribozyme.
16. "*
Murphy FL, Cech TR: GAAA tetraloop and conserved bulge stabilize tertiary structure of a group I intron domain. I Mol Biol 1994, 236:49-63. See annotation [18"']. Jaeger L, Michel F, Westhof E: Involvement of a GNRA tetraloop in long-range RNA tertiary interadions. J Mol Biol 1994, 236:1271-1276. This paper and [17•] define the helix-docked tetraloop motif, which is a long-range RNA tertiary interaction that contributes to the three-dimensional organization of many different RNA molecules. This general structure is of fundamental importance in RNA tertiary folding. Pley HM, Flaherty KM, McKay DB: Model for an RNA tertiary interaction from the structure of an inlermolecular complex between a GAAA tetraloop and an RNA helix. Nature 1994, 372:111-113. A high-resolution analysis of the helix-docked tetraloop motif, provided by the crystal structure of the hammerhead ribozyme. Chang K-Y, Tinoco I: Characterization of a "kissing" hairpin complex derived from the HIV genome. Proc Nat/ Acad Sci USA 1994, 91:8705-88709. This paper shows that the loop nucleotides of two hairpin RNAs can associate and drive the formation of a two-hairpin complex. 21. •
Pan T: Higher-order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry 1995, 34:902-909. See annotation [22*].
22. •
Pyle AM, Green JB: Building a kinetic framework for group II intron ribozyme activity: quantitation of interdomain binding and reaction rate. Biochemistry 1994, 33:2716-2725. This paper and [21 +} describe the assembly of large ribozymes from separate RNA domains, which form active complexes that can be studied using enzymological approaches. In this way, information about the tertiary architecture and interactions important to RNase P and group II intron folding was determined.
Malhotra A, Tan RKZ, Harvey SC: Modeling large RNAs and ribonucleoprotein parlicles using molecular mechanics techniques. Biophys J 1994, 66:1777-1795. An excellent discussion of current approaches and considerations important in the computer modeling of complex RNA structures.
35. "
Tuschl T, Gohlke C, Jovin TM, Westhof E, Eckstein F: A threedimensional model for the hammerhead ribozyme based on fluorescence measurements. Science 1994, 266:785-789. This meticulous application of fluorescence resonance energy transfer (FRET), combined with molecular modeling procedures, demonstrates the power of FRET for solution structural studies of RNA. The study provides an interesting comparison with the crystal structure of the ribozyme. 36. •
Woisard A, Fourrey J-L, Favre A: Multiple folded conformations of a hammerhead ribozyme domain under cleavage conditions. J Mol Biol 1994, 239:366-370. In light of various structural models for the hammerhead ribozyme [33"~,34•,35"] this work gives a physical description of conformational heterogeneity in the molecule.
23. Laing LG, Gluick TC, Draper DE: Stabilization of RNA structure " by Mg ions. J Mol Biol 1994, 237:577-587. A remarkable analysis of the three different roles that metal ions can play in the stabilization of RNA tertiary structure.
37. •
24. •
38. •
Yue D, Kintanar A, Horowitz J: Nucleoside modifications stabilize Mg 2+ binding in E. coli tRNAval: an imino proton NMR investigation. Biochemistry 1994, 33:8905-8911. Post-transcriptional base modifications were not previously thought to play a large role in the folding of tRNA; however, this study shows that modifications can be involved in the binding of stabilizing metal ions. 25.
26. *"
Arnez JG, Steitz TA: Crystal structure of unmodified IRNA GIn complexed with glulaminyl-tRNA synthelase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry 1994, 33:7560-7567.
Westhof E, Ahman S: Three-dimensional working model of Mt RNA, the catalytic RNA subunit of ribonuclease P from E. coli. Proc Nat/ Acad 5ci USA 1994, 91:5133-5137. See annotation [38"].
Harris ME, Nolan JM, Malhotra A, Brown JW, Harvey SC, Pace NR: Use of photoaffinity crosslinking and molecular modeling to analyze the global archileclure of ribonuclease P RNA. EMBO J 1994, 13:3953-3963. This paper and [37*] describe three-dimensional models of RNase P generated by the use of biochemical and phylogenetic techniques combined with computational approaches to RNA tertiary structure modeling. 39.
Mattson JG, Svard SG, Kirsebom LA: Charaderization of the Borrelia burgdorferl RNase P RNA gene reveals a novel tertiary interaction. J Mol Biol 1994, 241:1-6.
40.
Franzen JS, Zhang M, Chay TR, Peebles CL: Thermal activation of a group II inlron rihozyme reveals multiple conformational states. Biochemistry 1994, 33:11315-11326.
41. •"
Wang YH, Murphy FM, Cech TR, Griffith JD: Visualization of a tertiary structural domain of the Tetrahymena group I intron by electron microscopy. J Mol Biol 1994, 236:64-71.
Kowalak JA, Dalluge JJ, McCIoskey JA, Stetter KO: The
role of posttranscriplional modification in stabilization of transfer RNA from hyperthermoflbiles. Biochemistry 1994, 33:7869-7876. By exploring the role of base modifications in the folding of tRNAs from thermophitic organisms, the authors make important observations about the basis for thermodynamic stabilization and the folding pathways of RNA molecules.
309
31 0
Nucleic acids Mutagenesis and electron microscopy are combined, revealing a dramatic kinked conformation essential for activity of a group I intron structural domain.
42.
Burd CG, Dreyfuss G: Conserved structures and diversity of functions of RNA-binding proteins. Science 1994, 265:615-621. A useful reference for understanding the emerging field of RNA-protein interactions. ,
43. ""
Mohr G, Caprara MG, Guo Q, Lambowitz AM: A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme. Science 1994, 370:147-150. This study establishes an important new concept by demonstrating that a protein, provided in trans, can substitute for an essential RNA substructure in the Tetrahymena ribozyme. In so doing, the study underscores parallels in molecular recognition by RNA and proteins. 44. ""
Hall KB: Interaction of RNA hairpins with the human UIA N-terminal RNA binding domain. Biochemistry 1994, 33:10076-10088. See annotation [45ee]. 45. ""
48. ""
Herschlag D, Khosla M, Tsuchihashi Z, Karpel RL: An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis. EMBO J 1994, 13:2913-2924. Just as proteins can act as chaperones for protein folding, so can they also chaperone RNA folding, by selectively stabilizing particular RNA conformations. The concept is illustrated in this study of the effects of nucleocapsid protein on the kinetics of folding of hammerhead ribozymes. 49. •
Laggerbauer B, Murphy FL, Cech TR: Two major tertiary folding transitions of the Tetrahymenacatalytic RNA. EMBO J 1994, 13:2669-2676. See annotation [50ee]. 50. Zarrinkar PP, Williamson JR: Kinetic intermediates in RNA ee folding. Science 1994, 265:918-924. The folding pathway of the Tetrahymenaribozyme is described in this paper and [49"]. This paper is of considerable interest because it shows that the folding of a single RNA structural domain limits the rate of folding for the entire ribozyme. 51. ""
Laing LG, Draper DE: Thermodynamics of RNA folding in a conserved ribosomal RNA domain. J Mol Biol 1994, 237:560-576. See annotation [52e"].
Oubridge C, Ito N, Evans PR, Teo C-H, Nagai K: Crystal structure at 1.92A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature 1994, 372:432--438. Together, crystallographic studies (this paper) and NMR studies [44 e'] of RNA binding to the U1A protein provide a clear view of an RNA-protein interface, which is one of the first (other than the synthetase-tRNA interaction) to have been characterized.
52. Gluick TC, Draper DE: Thermodynamics of folding a pseudo•e knotted mRNA fragment. J Mol Biol 1994, 241:246-262. Together, this paper and [51"'] show that different global folding pathways will be adopted by different RNA molecules. They both show that the folding of secondary and tertiary structural elements can be interdependent.
46.
53.
47. e.
Nureki O, Niimi T, Muramatsu I, Kanno H, Kohno T, Florentz C: Molecular recognition of the identity-determinant set of isoleucine transfer RNA from E. coil ] Mol Biol 1994, 236:71 0-724.
Tan R, Frankel AD: Costahilization of peptide and RNA structure in an HIV Rev peplide-RRE complex. Biochemistry 1994, 33:14579-14585. The authors provide evidence that RNAs and proteins can co-fold, a concept that unites the respective problems of RNA and protein folding.
Cech TR, Damberger SH, Gutell RR: Representation of the secondary and tertiary structure of group I inlrons. Nature Struct Biol 1994, 1:273-280.
AM Pyle and JB Green, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.