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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.
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