In the fluorescent spotlight: global and local conformational changes of small catalytic RNAs

RNA is a ubiquitous biopolymer that performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. RNA is unique in that it can carry both genetic information and catalytic function. Its secondary structure domains, which fold stably and independently, assemble hierarchically into modular tertiary structures. Studies of these folding events are key to understanding how catalytic RNAs (ribozymes) are able to position reaction components for site-specific chemistry. We have made use of fluorescence techniques to monitor the rates and free energies of folding of the small hairpin and hepatitis delta virus (HDV) ribozymes, found in satellite RNAs of plant and the human hepatitis B viruses, respectively. In particular, fluorescence resonance energy transfer (FRET) has been employed to monitor global conformational changes, and 2-aminopurine fluorescence quenching to probe for local structural rearrangements. In this review we illuminate what we have learned about the reaction pathways of the hairpin and HDV ribozymes, and how our results have complemented other biochemical and biophysical investigations. The structural transitions observed in these two small catalytic RNAs are likely to be found in many other biological RNAs, and the described fluorescence techniques promise to be broadly applicable.     

Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.     

A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication.

The ribozymes of hepatitis delta virus (HDV) have so far been studied primarily in vitro. Several structural models for HDV ribozymes based on truncated HDV RNA fragments, which are different from the hammerhead or the hairpin/paperclip ribozyme model proposed for plant viroid or virusoid RNAs, have been proposed. Whether these structures actually exist in vivo and whether ribozymes actually function in the HDV replication cycle have not been demonstrated. We have now developed an in vivo ribozyme self-cleavage assay capable of detecting self-cleavage of dimer or trimer HDV RNA in vivo. By site-directed mutagenesis and compensatory mutations to disrupt and restore potential base pairing in the ribozyme domain of the full-length HDV RNA according to the various structural models, a close correlation between the detected in vivo and the predicted in vitro ribozyme activities of various mutant RNAs was demonstrated. These results suggest that the proposed in vitro ribozyme structure likely exists and functions during the HDV replication cycle in vivo. Furthermore, the pseudoknot model most likely represents the structure responsible for the ribozyme activity in vivo. All of the mutants that had lost the ribozyme activity could not replicate, indicating that the ribozyme activities are indeed required for HDV RNA replication. However, some of the compensatory mutants which have restored both the cleavage and ligation activities could not replicate, suggesting that the ribozyme domains are also involved in other unidentified functions or in the formation of an alternative structure that is required for HDV RNA replication. This study thus established that the ribozyme has important biological functions in the HDV life cycle.     

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2′-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape.     

Helical junctions as determinants for RNA folding: origin of tertiary structure stability of the hairpin ribozyme

Helical junctions are ubiquitous structural elements that govern the folding and tertiary structure of RNAs. The tobacco ringspot virus hairpin ribozyme consists of two helix-loop-helix elements that lie on adjacent arms of a four-way junction. In the active form of the hairpin ribozyme, the loops are in proximity. The nature of the helical junction determines the stability of the hairpin ribozyme tertiary structure [Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Nat. Struct. Biol. 6, 544-549] and thus its catalytic activity. We used two-, three-, and four-way junction hairpin ribozymes as model systems to investigate the thermodynamic basis for the different tertiary structure stabilities. The equilibrium between docked and extended conformers was analyzed as a function of temperature using time-resolved fluorescence resonance energy transfer (trFRET). As the secondary and tertiary structure transitions overlap, information from UV melting curves and trFRET had to be combined to gain insight into the thermodynamics of both structural transitions. It turned out that the higher tertiary structure stability observed in the context of a four-way junction is the result of a lower entropic cost for the docking process. In the two- and three-way junction ribozymes, a high entropic cost counteracts the favorable enthalpic term, rendering the docked conformer only marginally stable. Thus, two- and three-way junction tertiary structures are more sensitive toward regulation by ligands, whereas four-way junctions provide a stable scaffold. Altogether, RNA folding and stability appear to be governed by principles similar to those for the folding of proteins.     

小型核酶的结构和催化机理

自然界存在的小型核酶主要有锤头型核酶、发夹型核酶、肝炎δ病毒（HDV）核酶和VS核酶.锤头型核酶由3个短螺旋和1个广义保守的连接序列组成;发夹型核酶的催化中心由两个肩并肩挨着的区域构成;HDV核酶折叠成包含五个螺旋臂（P1～P4）的双结结构;VS核酶由五个螺旋结构组成,这些螺旋结构通过两个连接域连接起来.小型核酶的催化机理与其分子结构密切相关.金属离子或特定碱基都可作为催化反应的关键成分.锤头型核酶的催化必须有金属离子（尤其是二价金属离子）参与,而发夹型核酶则完全不需要金属离子.基因组HDV核酶进行催化时要有金属离子和特定碱基互相配合.     

Single-molecule Förster resonance energy transfer studies of RNA structure, dynamics and function

Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.     

Importance of short pseudoknot base pairs between two single-stranded regions of HDV ribozyme.

Human hepatitis delta virus (HDV) ribozyme can catalyze self-cleavage reaction in the presence of Mg2+ ions, yielding products with 2',3'-cyclic phosphate and 5'-OH termini as do hammerhead and hairpin ribozymes. Recently, the tertiary structure of 3'-cleaved product of genomic HDV ribozyme was solved by X-ray crystallographic analysis. In this structure three single-stranded regions (SSrA, -B and -C) interacts intricately with hydrogen bonds between bases, phosphate oxygens and 2'-OHs to form nested double pseudoknot structure. Especially two Watson-Crick base pairs, 726G-710C and 727G-709C, between SSrA and SSrC, seems to be important for compact folding. To characterize the necessity of the two base pairs, we performed in vitro selection of active ribozymes using random RNA pool which mutated at 709, 710, 726 and 727. The result indicates that basically one G-C base pair is necessary for the activity.     

Energetics of hydrogen bond networks in RNA: hydrogen bonds surrounding G+1 and U42 are the major determinants for the tertiary structure stability of the hairpin ribozyme

The hairpin ribozyme, a small catalytic RNA consisting of two helix-loop-helix motifs, serves as a paradigm for RNA folding. In the active conformer, the ribozyme is docked into a compact structure via loop-loop interactions. The crystal structure of the docked hairpin ribozyme shows an intricate network of hydrogen bonding interactions at the docking interface, mediated by the base, sugar, and phosphate groups of U42 and G+1 [Rupert, P. B., and Ferre-D'Amare, A. R. (2001) Nature 410, 780-786]. To elucidate the determinants for tertiary structure stability in the hairpin ribozyme, we evaluated the energetic contributions of hydrogen bonds surrounding U42 and G+1 by time-resolved fluorescence resonance energy transfer using modified ribozymes that lack one or more of the individual interactions. Elimination of a single tertiary hydrogen bond consistently resulted in a net destabilization of approximately 2 kJ/mol. The results of double- and triple-mutant cycles suggest that individual hydrogen bonds surrounding G+1 or U42 act cooperatively and form extended hydrogen bond networks that stabilize the docked ribozyme. These results demonstrate that RNAs, similar to proteins, can exploit coupled hydrogen bond networks to stabilize the docking of distant structural domains.     

Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis

The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the double-rolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C(75)) with a pK(a) close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5'-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G(76) of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP(76) is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP(76) becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C(75) becomes positioned for reaction chemistry only along the trajectory from precursor to product.     

Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.     

Combinatorial Screening and Intracellular Antiviral Activity of Hairpin Ribozymes Directed against Hepatitis B Virus

A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.     

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme.

Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme.     

Sequence and structure of the catalytic RNA of hepatitis delta virus genomic RNA.

Human hepatitis delta virus (HDV) RNA has been shown to contain a self-catalyzed cleavage activity. The sequence requirement for its catalytic activity appears to be different from that of other known ribozymes. In this paper, we define the minimum contiguous sequence and secondary structure of the HDV genomic RNA required for the catalytic activity. By using nested-set deletion mutants, we have determined that the essential sequence for the catalytic activity is contained within no more than 85 nucleotides of HDV RNA. These results are in close agreement with the previous determinations and confirmed the relative insignificance of the sequence at the 5' side of the cleavage site. The smallest catalytic RNA, representing HDV genomic RNA nucleotide positions 683 to 770, was used as the basis for studying the secondary structure requirements for catalytic activity. Analysis of the RNA structure, using RNase V1, nuclease S1 and diethylpyrocarbonate treatments showed that this RNA contains at least two stem-and-loop structures. Other larger HDV RNA subfragments containing the catalytic activity also have a very similar secondary structure. By performing site-specific mutagenesis studies, it was shown that one of the stem-and-loop structures could be deleted to half of its original size without affecting the catalytic activity. In addition, the other stem-and-loop contained a six base-pair helix, and the structure, rather than the sequence, of this helix was required for the catalytic activity. However, the structure of a portion of the stem-and-loop remains uncertain. We also report that this RNA can be divided into two separate molecules, which alone did not have cleavage activity but, when mixed, one of the RNAs could be cleaved in trans. This study thus reveals some features of the secondary structure of the HDV genomic RNA involved in self-catalyzed cleavage. A model of this RNA structure is presented.     

Self-cleavage of a 71 nucleotide-long ribozyme derived from hepatitis delta virus genomic RNA.

Self-cleavage efficiency of ribozymes derived from hepatitis delta virus (HDV) has been shown to depend on the RNA structure, which in turn may be determined by the length of the considered sequences. Here we describe the construction and functional analysis of a 71 nucleotide-long RNA genomic fragment, Rz71, which carries an 18 nucleotide deletion in a very stable GC-rich stem-loop (stem IV), predicted to be present in several computer-derived secondary structure models. Rz71 is able to undergo self-cleavage under non-denaturing conditions (the t1/2 of the reaction at 37 degrees C is 3 min). The deletion, however, is not neutral, since under the same conditions the non-deleted ribozyme cleaves to 50% in less than 15 sec. Therefore, stem-loop IV seems to play a structural role, not being directly involved in the catalytic reaction, but contributing to the correct positioning of the catalytic core of the HDV ribozyme. Rz71 is the smallest self-cleaving sub-fragment of HDV genomic RNA reported so far.     

RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency.     

Pseudoknots: a new motif in the RNA game.

In the last few years a novel RNA folding principle called pseudoknotting has emerged. Originally discovered in noncoding regions of plant viral RNAs, pseudoknots now appear to be a widespread structural motif in a number of functionally different RNAs. These structural elements are part of tRNA-like structures and are involved in folding catalytic sites of ribozymes. They increase the efficiency of ribosomal frameshifting or can serve as specific binding sites for regulatory proteins.