Funding to Students:
- NIH NRSA Predoctral Award (NIH F31-NS054492) to Josh Sokoloski
Funding to PCB:
- National Science Foundation (NSF MCB-0527102) "The Influence of Secondary Structure on the Folding and Catalysis of Functional RNAs" Bevilacqua (PI)
- National Institutes of Health (NIH R01-GM58709) "HDV RNA Folding and PKR Protein Regulation" Bevilacqua (PI)
- National Science Foundation (NSF MCB-0345251) "Roles of the Arabidopsis AKIP RNA-binding Proteins in Guard Cell Function" Sally Assmann (PI)/ Bevilacqua (co-PI)
We use RNA and DNA oligonucleotides to better understand the behavior of RNA folding and ligand binding in larger functional RNAs. The local interactions in RNA--stacking, hydrogen bonding, and electrostatics--are strong. As such, RNA tends to fold in a largely hierarchical fashion, with secondary structures folding independently of tertiary structures, and tertiary structures being dependent on the presence of secondary structure to fold. This characteristic of RNA folding allows us to take a "divide-and-conquer" approach, studying the properties of independently stable secondary structural elements. There are a number of important applications of these studies, including providing free energy parameters to RNA structure prediction algorithms.
One of the contributions our lab has made in this area is developing a combinatorial approach to thermodynamics. Because of the larger size of the motifs not yet explored in RNA folding, we applied in vitro selection (or SELEX) methods and temperature gradient gel electrophoresis (TGGE) to the stability of RNA and DNA folding. This work is funded by the NSF in the form of a Career Award. In this approach, all possible sequences are prepared at once and only the most interesting--in this case the most or least stable--are isolated, identified, and characterized. This has led to the identification of new and functionally important hairpin loops in RNA and DNA of various sizes (17, 29, 31). This work involves a collaboration with Amy Parente at Penn State, Altoona--a primarily undergraduate institution.
These studies led to the identification of some new behaviors and approaches for studying oligonucleotides. In collaboration with Ryszard Kierzek at the Polish Academy of Sciences, we showed that restricting the conformation of RNA with modified oligonucleotides, such as the syn base 8Br-guanosine, improves the conformational uniformity of RNA (33, 48). We then went on to study the consequences of conformational restriction on the kinetics of RNA folding, in which enhanced stability was attributed to faster folding rather than slower unfolding (48). This work involves the rapid kinetics method of temperature jump (T-jump), and is part of an ongoing collaboration with Martin Gruebele at UIUC.
We are especially interested in understanding cooperativity in the folding of small RNAs. We have shown that small, stable DNA loops fold in a highly cooperative, all-or-none-like, fashion, while RNA loops are not as cooperative. The molecular basis for this is the extra hydrogen bonding in RNA. This work indicates that caution should be used in interpreting free energy penalties from functional group deletions in terms of individual hydrogen bonds (32, 40, 43, 44).
Most recently, we are making progress in bridging the gap between oligonucleotide studies and ribozyme studies (described in next section). In particular, we recently developed a simple and efficient way to determine pKa values in RNA by modifying the backbone with a phosphorothioate (46). Current efforts are directed at connecting pKa shifting and cooperativity of RNA folding (51, 53) in collaboration with Juliette Lecomte. pKa shifts towards neutrality are of interest because they provide a potential way to increase the functional diversity of RNA (41).
2.) Ribozyme Folding and Chemistry
In the early 1980's, Tom Cech and Sidney Altman showed that RNA could be an enzyme--a so-called 'ribozyme'-- catalyzing the making and breaking of covalent bonds. This led to the 1989 Nobel Prize in Chemistry. The Bevilacqua group is interested in answering two fundamental questions about ribozymes: 1.) How do they fold into their functional structures?, and 2.) What are the chemical mechanisms that ribozymes use to achieve catalysis?
Regarding folding, we have focused mostly on the small (ca. 85 nt) self-cleaving ribozyme from the hepatitis delta virus (HDV). This ribozyme is an attractive candidate for study because of its relevance to human health as a pathogen. Although this is a relatively small RNA, it has a complex topology and folding pathway. It consists of two nested pseudoknots and is prone to adopting misfolded structures that are incapable of carrying out phosphodiester bond cleavage. We have worked to identify the role of sequence flanking the ribozyme in mediating bond cleavage, as well as ribozyme sequence itself (20, 26, 30). Recently, we showed that non-native structures can act as 'folding guides' and actually facilitate native ribozyme folding in some instances (45). Work continues in the lab to uncover the importance of folding during transcription, as well as the importance of RNA-protein interactions in chaperoning folding of the ribozyme.
There are several crystal structures of the HDV ribozyme, and they have implications for the involvement of the nucleobases in catalysis. In particular, C75 appears to be important in the reaction mechanism. Kinetic ambiguity prevents simple determination of the role of C75 as the general acid or base in the reaction (34). We have carried out kinetics experiments that support a general acid role for C75 (19, 24, 25, 35). Current work in the lab is directed towards deepening our understanding of the mechanism through structure-function studies.
In the cell, RNA is typically associated with proteins. A large number of RNA-protein interactions are known. We are studying two RNA-protein interactions in our lab, and several others through collaborations. PKR is the RNA-activated protein kinase and mediates an anti-viral response in humans. PKR is activated by long stretches of perfect dsRNA to autophosphorylate and then phosphorylate eIF-2a, thereby inhibiting the initiation of translation. We have shown that the dsRNA binding domain (dsRBD), which consists of two dsRBMs (reviewed in 49), has a site size of about 16 bp and that PKR is activated by non-canonical RNA structures (12, 13). Recently, we demonstrated that the PKR can be activated by short dsRNAs, as long as they have single-stranded tails (50), and that the dsRBD is capable of modulating the topology of RNA by straightening bent RNA (21, 22). Current work on this project is aimed at understanding, at the molecular level, the elements of the RNA responsible for activation of PKR, as well as mechanistic characterization of the activation process by canonical and non-canonical RNA motifs, especially those in the HDV RNA.
We also have two active collaborations on RNA-protein interactions with research groups at Penn State. We are working with Paul Babitzke in the BMB department to understand the role of the TRAP protein in mediating tryptophan biosynthesis in Bacillus. This work involves a number of intriguing RNA conformational changes that regulate tryptophan production at both the transcriptional and translational levels. This work involves collaboration on biochemical and biophysical structure-function studies, as well as in vivo prokaryotic gene expression studies in Paul's lab. We have published several papers (37-39), and graduated one Ph.D student.
Our other Penn State collaboration on RNA-protein interactions (NSF funded) is with Sally Assmann in the Biology department. We are working to understand how the plant hormone abscisic acid (ABA) allows plants to acclimate to environmental stresses such as drought and cold, which is critical to crop productivity. ABA appears to have important roles in RNA metabolism. The role of three RNA binding proteins (AtAKIPs) in this process are being studied, including determining RNA targets in vivo through a ribonomics approach.