Folding and assembly of ribonucleoprotein (RNP) enzymes
1. Structural dynamics and enzymatic activity of the hairpin ribozyme (past achievement)
2. Single-molecule transition-state analysis (past achievement)
3. Protein-dependent RNA folding - bI5 group I intron (past achievement and current project)
4. Folding and assembly of complex RNP enzymes - archaeal ribonulease P (current project)
5. Assembly and function of telomerase (current project)
1. Structural dynamics and enzymatic activity of the hairpin ribozyme (past achievement)
We have studied the conformational dynamics of RNA enzymes by imaging single RNA molecules in real time using f luorescence resonance energy transfer (FRET). FRET is a powerful assay to measure the structural dynamics of biomolecules in real time. In this assay, a pair of fluorescent donor and acceptor is attached to the host biomolecule of interest. The energy transfer efficiency between the donor and acceptor depends on their intermolecular distance, making FRET sensitive to the conformational change of the host molecule (Fig. 1, Movie 1). Our experiments have revealed transient states and multiple kinetic paths that are difficult to detect by classical ensemble experiments , demonstrating the power of single-molecule FRET to elucidate the complex conformational dynamics of RNA. Indeed, our previous experiments on the Tetrahymena ribozyme show that this large RNA enzyme folds along multiple pathways and traverse intermediate states before attaining their native structure.
Figure 1. A FRET scheme and experimental time-traces showing anticorrelated changes in the donor and acceptor signals, indicating comformational change of the host molecule.
Using single-molecule FRET, we have characterized the structural dynamics and enzymatic reaction of the hairpin ribozyme, a small RNA enzyme. This first-of-a-kind experiment demonstrated the power of single-molecule methods for detecting transient intermediate states, and thereby resolving microscopic steps of a complex reaction. Following the reaction of single ribozyme molecules, we have shown that the hairpin ribozyme catalyzes the cleavage reaction of its substrate in several steps: (i) substrate binding to the ribozyme; (ii) folding of the ribozyme-substrate complex into a catalytically active state (docking); (iii) cleavage of the substrate; (iv) unfolding of the ribozyme-product complex (undocking); and (v) release of the cleavage products (Fig. 2). Our work has provided important insights into RNA enzymology. By determining the rate constants of each microscopic step, we found that the overall cleavage reaction of the two-way-junction ribozyme is primarily rate-limited by the two structural transition steps, (ii) and (iv). Considering that RNA molecules tend to have slow structural dynamics, this could be a general rate-limiting mechanism for RNA enzymatic reactions.
Figure 2. A single molecule investigation of the hairpin ribozyme reaction pathway. (A) The 2-way junction form of the ribozyme. The Cy3 and Cy5 dyes serve as the fluorescent donor and acceptor. (B) Following the structural dynamics and function of single ribozyme molecules. (Upper and middle panels) A fluorescence time trace of a single ribozyme that shows binding of a substrate, docking and undocking. Green and red lines are the fluorescence signals from the donor and acceptor, respectively. The blue line is the corresponding FRET value. The substrate-free ribozyme gives FRET ~ 0.4. Upon binding of a substrate, the FRET time-trace exhibits stochastic transitions between two levels (0.2 and 0.8) that correspond to the undocked and docked states, respectively. (Lower panel) FRET time-trace of a single complex of the ribozyme showing docking, undocking and cleavage. The release of products after cleavage recovers the ribozyme to a substrate-free state with FRET ~ 0.4. (C) The reaction pathway of the hairpin ribozyme. Rz, S, 3'P and 5'P stand for the ribozyme, the substrate, the 3' and 5' cleavage products, respectively.
Taking advantage of the capability of the single-molecule approach to fully dissect the reaction pathway (Fig. 2), we have investigated the effects that modifications of essential functional groups have on individual rate constants on the reaction path. Surprisingly, even modifications remote from the catalytic site can directly affect the internal chemistry (cleavage and ligation) rate constants, suggesting a network of coupled molecular motions connecting distant parts of the RNA with its reaction site.
Most strikingly, we have found that even a small RNA enzyme have rather complex, heterogeneous structural dynamics. Single-molecule time traces indicate that the ribozyme can assume several distinct docked and undocked states that slowly interconvert under functional conditions (Fig. 3). Such heterogeneous structural dynamics appears to be general and are observed in all variant hairpin ribozymes that we have studied. These results indicate a highly rugged energy landscape in the conformational space of the RNA molecule.
Figure 3. Heterogeneous conformational dynamics of the hairpin ribozyme. (A) The FRET time-traces of individual ribozyme molecules show heterogeneous undocking kinetics with dramatically different rate constants. The distributions of dwell times of undocked and docked states indicate that the hairpin ribozyme docks with a single rate constant but undocks with at least four different rate constants , each exemplified by a trace shown here. The molecules also retain m emory of their undocking kinetics for many hours. (B) Examples of memory loss after 3 hours.
Movie 1. Visualizing the conformational transitions of individual ribozyme molecules. Each bright sport is the fluorescence signal of a single ribozyme molecule. The fluorescence image is false color such that high FRET value is depicted in red and low FRET is in green.
Reference:
- X. Zhuang, H. Kim, M. Pereira, H. Babcock, N. Walter, S. Chu, " Correlating structural dynamics and function in single ribozyme molecules ," Science 296 , 1473-1476 (2002).
- D. Rueda, G. Bokinsky, M. M. Rhodes, M. J. Rust, X. Zhuang, N. G. Walter, " Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance ," Proc. Natl. Acad. Sci. USA 101 , 10066-10071 (2004).
- X. Zhuang , L. Bartley, H. Babcock, R. Russell, T. Ha, D. Herschlag, S. Chu, " A single-molecule study of RNA catalysis and folding ," Science 288 , 2048-2051 (2000)
2. Single-molecule transition state analysis (past achievement)
Characterization of the transition state (ensemble) is critical for a mechanistic understanding of the macromolecule-folding problem. While transition-state analysis has been performed routinely for protein folding, its implementation in RNA folding is challenging due to the rugged energy landscape of RNA, which often leads to the coexistence of distinct structural transitions. Recognizing the power of single-molecule measurements to isolate individual transitions, we developed single-molecule phi-value analysis to determine the degree to which specific intramolecular interactions are formed in the transition states of RNA folding.
Using FRET to monitor the conformational changes of individual RNA molecules, in conjunction with site-specific mutations, we characterized the folding transition states of the hairpin ribozyme and found these states to have a compact overall topology, but without substantially formed native tertiary contacts (Fig. 4). The compact transition states, having lower conformational entropy than that of the unfolded states, could present a bottleneck in the folding reaction. The generality of such transition states may extend to other biopolymers - recent studies have shown that some proteins also feature folding transition states with a native-like topology that lack native tertiary contacts.
Figure 4. Transition state analysis of the hairpin ribozyme. (A) Effect of mutations on the rate constants for docking and undocking. The dG11, dC12 and a+1:U25 mutations destabilize the three sets of tertiary interactions formed in the docked state of the ribozyme. (B) The F -values of the above modifications. (C) Dependence of k dock and k undock of the wild-type 2-way junction ribozyme on Mg 2+ concentration. (D) Free energy diagram of the docking and undocking transitions.
Reference
3. Protein-dependent RNA folding - bI5 group I intron (past achievement and current project)
The above results, together with experiments by other investigators, suggest that RNA folds across a highly rugged energy landscape, which not only dictates the structural dynamics but also has functional consequences - the catalytic reactions of RNA can be severely rate-limited by their slow conformational transitions. This problem is mitigated in nature by the association of RNA with protein cofactors to form RNP complexes. Indeed, most RNA molecules fold with the help of proteins. How proteins assist RNA folding is, however, not well understood. Our RNA research currently focuses on the folding and assembly of RNP complexes to 
address the following important questions: what are the critical factors that limit the structural transitions in RNA, and how do proteins affect these factors.
We have begun to investigate a protein-dependent RNA enzyme, the self-splicing group I intron bI5, primarily using single-molecule FRET (Fig. 5). This RNP is made of one RNA (bI5) and one protein (CBP2) component. Our preliminary studies show that in addition to stabilizing the native structure of bI5, the protein cofactor CBP2 helps the bI5 RNA to traverse many conformations not otherwise accessible to the RNA. The RNA-protein complex folds along multiple pathways with distinct intermediate states and folding rate constants. We are currently characterizing the structural properties of these intermediates and pathways, to provide a detailed picture of the folding dynamics and equilibrium conformational dynamics of this RNP and to elucidate the molecular mechanisms by which protein facilitate RNA folding.
We will extend our study to include other proteins that are known to assist RNA folding and subsequently to other RNA systems, aiming to obtain a broader understanding of the molecular mechanisms by which proteins facilitate RNA folding.
Figure 5. The bI5 ribozyme and one of the labeling
scheme that we used for FRET analysis.
4. Folding and assembly of complex RNP - archaeal ribonulease P (current project)
In keeping with our progressive approach, we are take our investigation to the next level of complexity by studying the assembly of complex RNP enzymes made of several components. The archaeal ribonuclease P (RNase P), a complex RNP responsible for the 5'-end maturation of tRNA, is one such example. This RNP holoenzyme contains one RNA molecule and four proteins. While one or more of the proteins may facilitate the binding and dissociation of tRNA substrates, most of the protein cofactors are believed to be important for assisting RNA folding and RNP assembly. In vitro reconstitution of the active holoenzyme was recently accomplished by our collaborators. The effect of each protein component on the RNA structure and on the binding of other proteins is, however, unknown.
U sing single-molecule FRET with different fluorescent-labeling schemes, in conjunction with site-specific mutations, we plan to monitor the conformational dynamics of the RNase P RNA and to analyze how they are affected by the protein components of RNase P. By labeling these proteins and using fluorescence colocalization, we aim to determine the assembly sequence of individual protein components. These single-molecule experiments will be complemented by classical biochemical and structural characterizations that can provide static, structural information of this RNP complex with higher spatial resolution (being carried out in our collaborators' labs ) . Our final goal is to obtain, using this combined approach, a "molecular movie" of the assembly process of this RNP complex and a mechanistic picture of its biogenesis at the molecular level. This approach should be applicable to other RNP complexes.
5. Assembly and function of telomerase (current project)
Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of linear chromosomes, thereby promoting genomic stability. The addition of short DNA repeats by telomerase circumvents replication-dependent telomere attrition. Telomerase is reactivated in approximately 85%-90% of human cancers, and the potential of telomerase drug targeting has been demonstrated. The enzymatic activity of the telomerase RNP requires both the telomerase RNA and the telomerase reverse transcriptase (TERT). A telomerase holoenzyme protein (p65) was also shown to facilitate RNP assembly in vivo.
Using single-molecule FRET, we are investigating the conformational dynamics of the telomerase RNA, the effect of P65 and TERT on the RNA conformation and the assembly process of the telomerase RNP. Next, we shall study Telomerase structural dynamics during the telomerase catalytic cycle. Combining single molecule FRET and magnetic tweezers, we will analyze the mechanochemical properties of the telomerase machine.
Our collaborators on the folding and assembly of ribonucleoprotein enzymes:
Links to our other research areas:
-
Cellular entry of viruses and non-viral gene delivery vectors
-
Developing new techniques for single-molecule detection and bio-sensing
- Return to Research