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RNA

 
 

There are several reasons for RNA as a fascinating subject for study: importance for biology, dearth of information extant, applicability of NMR given recent methodological and labeling developments, and (perhaps most important) challenge. Collaborative studies on a few different systems are underway.
Replication of human immunodeficiency virus type-1 (HIV-1) entails packaging of two identical, noncovalently linked copies of full length genomic RNA into each virion. This 'dimer packaging' is governed by specific sequences and their interaction with the Gag polyprotein. The RNA strands first interact by base-pairing via a six-base palindrome in the loop of a stem-loop, SL1. We have solved the structure of the intermediate loop-loop homodimer formed by a truncated version of SL1. It presents an interface containing standard Watson-Crick base-pairing between two loop palindromes and quaternary interactions involving essential A's. This work is done in collaboration with Prof. Tris Parslow's lab.


Telomerase is a eukaryotic ribonucleoprotein that synthesizes telomeric DNA. The telomere occurs at the ends of chromosomes. Telomerase apparently also acts by capping telomeres, so that the terminal DNA will not appear as a break and thus be susceptible to DNA damage-response. Recently, in collaboration with Prof. Elizabeth H. Blackburn's lab, we used in vitro telomerase assays, NMR and ultraviolet absorbance melting analyses of model oligonucleotides, we described functional, structural and energetic properties of a crucial moiety of the hTR, human telomerase RNA. In particular, we demonstrated that this crucial RNA domain exists in two alternative states of nearly equal stability in solution: one is the previously proposed pseudoknot formed by pairing one RNA moiety (P3) with the loop domain of another RNA moiety (P2b), and the other is a structured P2b loop alone. We show that the two-base mutation present in one gene copy in a family with the disfiguring disease dyskeratosis congenita abrogates telomerase activity by stabilizing the P2b loop structure. Likewise, stabilizing the pseudoknot structure also strongly attenuates telomerase activity. Like RNAs of other complexes such as the spliceosome, telomerase RNA appears to be a dynamic molecule whose function requires it to assume more than one secondary conformation.


Enteroviruses, such as poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3), and rhinoviruses, are members of the picornaviridae family. These positive strand RNA viruses carry their genetic information on a small (<10,000 bases) single-stranded genomic RNA molecule. This genomic RNA serves at least three important functions: it acts as a mRNA for viral protein synthesis; it is used as a template for negative-strand RNA replication; it is also involved in the packaging of new virions. To fulfill the multiple functional roles of its genomic RNA, the virus must have evolved certain regulatory mechanisms to balance the usage of the genomic RNA in different stages of the viral life cycle. We have recently brought structural biology to bear in a conquer-and-divide approach that has as its goal the unveiling of the regulation of these mechanisms. Studies along these lines involve RNA structures in the 5'UTR and associated regulatory proteins.