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Aminoacyl-tRNA
Synthetases. Members
of this ancient family of proteins catalyze the esterification
of an amino acid onto the 3'-end of tRNAs, which translate
the genetic code through codon-anticodon recognition. Figure
1 depicts the evolutionary conserved 5¡Ç-monophosphate that
is critical for efficient aminoacylation by E. coli and S.
cerevisiae HisRS. By
virtue of their specific recognition of tRNAs and their capability
to discriminate among structurally similar amino acids, the
aminoacyl-tRNA synthetases play a central role in assuring
a high degree of accuracy in protein synthesis. Site-directed
and atomic group "mutagenesis" are common methods used in
the lab to probe tRNA recognition. The latter approach allows
us
to identify specific functional groups that contribute to
synthetase recognition of RNA substrates. Overuse of antibiotics
in combination
with increased numbers of immunocompromised patients has
led to a dramatic increase in the incidence of antibiotic
resistance.
The aminoacyl-tRNA synthetases are an essential family of
enzymes offering up to twenty targets per pathogen. With
this in mind,
we are probing species-specific differences in substrate
recognition for drug design and to gain insights into the
development of
aminoacylation systems through evolution. In comparison to
our understanding of tRNA discrimination, relatively little
is known about the mechanism synthetases use to discriminate
amongst structurally related amino acids. To correct misactivation
of noncognate substrates, many synthetases require editing
activity, and studies aimed at understanding the mechanism
of translational editing are also underway. Figure 2 demonstrates
a novel triple-sieve mechanism for ProRS editing that has
been proposed by our lab. One goal of this work is to
use our knowledge of
amino acid recognition
and
editing to engineer novel synthetase-tRNA pairs for the incorporation
of unnatural amino acids into proteins in vitro and in
vivo.
Figure 1: Evolutionary
conserved 5¡Ç-monophosphate is critical to efficient aminoacylation
by E. coli and S. cerevisiae HisRS
Figure 2: A Triple-sieve Mechanism for ProRS Editing
Nucleic Acid-Protein
Interactions in HIV. During
the life cycle of HIV, its RNA genome must be converted into DNA.
This conversion is catalyzed by reverse transcriptase, an enzyme
that uses a specific host cell tRNALys molecule
as a primer. The process by which HIV selects and uses a specific
primer tRNA is not well understood, but we have recently shown
that human lysyl-tRNA synthetase (LysRS) is also packaged into
HIV and appears to be a critical factor in specific tRNA packaging.
Ongoing work is aimed at elucidating the molecular interactions
between human LysRS and HIV proteins. Figure 3 illustrates the
propsed tRNA pacckaging complex for HIV-1. In
vivo, the
tRNA primer and the HIV RNA genome must be unwound and annealed
together
before reverse transcription can be initiated. The annealing process
is mediated by the HIV nucleocapsid protein (NC), a nucleic acid "chaperone" protein
that facilitates nucleic acid rearrangements (Figure 4). We have
reconstituted in vitro systems that closely mimic several steps
of the reverse
transcription process in HIV to elucidate the mechanism of NCs
chaperone funtion. Experimental approaches we are currently using
to elucidate key nucleic acid-protein interactions in this system
include fluorescence resonance energy transfer (FRET), chemical
footprinting, and single molecule DNA stretching.
Figure 3: Proposed HIV-1 tRNA Packaging Complex
Figure
4: HIV-1 Nucleocapsid Protein and its Two Functional Domains
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