Been Laboratory Research Interests
Interests in this lab are directed at understanding various aspects of RNA structure and function with a focus on catalytic RNAs. Most of the work currently is directed at developing a detailed understanding of the structure, dynamics and chemical mechanism of the self-cleaving RNAs sequences (ribozymes) in hepatitis delta virus (HDV).
Hepatitis delta virus is an unusual human pathogen; it is a satellite of hepatitis B virus and contains a small circular RNA genome. In humans, the symptoms of infection can be severe, and in populations where hepatitis B is endemic and supportive therapy is lacking, HDV infection represents a serious, life-threatening disease. Even where care is available, there is no clear course of therapy.
Our studies have focused on the structure and function of a key element in the replication of the virus; a self-cleaving sequence in the RNA which is responsible for processing replication products into viral size genomes. Our initial work was directed at establishing the conditions required for cleavage and the sequences required for optimal activity. This led us to propose a novel secondary structure for the RNA sequence several years ago. The original secondary structure was in good agreement with a recent crystal structure from the Doudna lab and, with minor modifications, the current version is thought to accurately describe several features required for cleavage activity. However, much remains unknown about structural changes that may occur in the course of the cleavage reaction. Understanding how the structure and possible changes in structure related to RNA cleavage activity remain a goal of the lab.
Until very recently, most ribozymes were thought to be “metallo-enzymes” in that divalent cations, required for activity, where thought to be primarily and directly responsible for the catalysis. Although a more direct involvement of RNA in catalysis was hypothesized, no examples were known and the chemical properties of RNA made its sugar, bases and phosphate groups poorly suited to function as catalysts. It is now apparent that some ribozymes should be considered metal-assisted enzymes since, at least for the HDV, RNA sidechains (nucleobases) are indeed functioning directly in acid-base catalysis. Recently, we have demonstrated that a cytosine side chain is acting directly in proton transfer in the rate determining step of the phosphotransesterification reaction that results in cleavage. We are currently defining the details of this reaction. It is significant that in the HDV ribozyme, substituting the catalytic cytosine with adenine results in an active mutant; from a recent crystal structure of the ribosome (a ribozyme) it has been proposed that an adenine is acting as a general-acid-base catalyst. Other catalytic strategies used by the HDV ribozymes include metal ion catalysis and reduction in the activation energy difference though ground-state detabilization upon substrate binding. The details of all these, and other unidentified strategies, remain to be defined but the HDV ribozymes turn out to be powerful model systems to study many features of RNA catalysis.