Transfer RNAs (tRNAs) are fundamental to cellular life. They have a major role in translation of genetic information, and in replication of RNA retroviruses. They may be also important for development of bacterial pathogenesis. The study of tRNAs and protein enzymes that recognize tRNAs are thus important to provide insights into the origins of the genetic code and to suggest new concepts and stategies that target tRNAs for medical applications.
1. tRNA structure and function in decoding.
The L-shaped tRNA tertiary structure is a hallmark of many RNA structures found in the ribosomal translational machinery. This L-shape is formed by joining the acceptor T-stem with the D-anticodon stem through extensive stacking and tertiary H-bonding interactions, possible only in RNA. While previously thought to be a scaffold of tRNA, emerging studies have now shown that the L-shape is important for tRNA decoding. This includes recognition of tRNA by aminoacyl-tRNA synthetases, by elongation factor Tu (EF-Tu), by transloaction factor (EF-G), and by the ribosome. The emphasis here is to develop single turn-over kinetic assays to determine how the tRNA L-shaped structure contributes to the specificity of decoding.
2. tRNA repair and maintenance of the CCA end.
Of the three major steps of genetic information transfer from DNA, to RNA, to protein, only the repair of DNA is well known and characterized. Because DNA is the repository of genetic information, repair of DNA is essential and it has been thought that damaged RNA and protein molecules are dispensable. However, recent studies show that our cellular machinery actually makes an effort to repair damaged tRNA. Further, the repair enzyme turns out to be the same as those that repair the DNA. Repair of RNA would be particularly useful for larger rRNAs. Here, using tRNA as the model molecule, the repair mechanism will be elucidated and enzyme better studied in the structure-function relationships. In addition, a separate repair mechanism is required for the CCA end, which is the site of amino acid attachment. The CCA end is repaired and synthesized by the CCA-adding enzyme, a fascinating enzyme that does not use a nucleic acid template for the synthesis. The mechanism of the un-templated CCA-synthesis is being studied by mapping nucelotide-binding and tRNA-binding sites, transient kinetics, and genetic analysis.
3. Specificity of aminoacyl-tRNA synthetases.
Aminoacyl-tRNA synthetases are responsible for attaching amino acids to tRNA molecules. The specificity of these enzymes is exquisite and must be excuted at both the amino acid and tRNA levels. A major focus is on cysteinyl-tRNA synthetase (CysRS), which discriminates against the closely similar serine by a factor of 108-fold without the need of an editing function. In collaboration with Dr. John Perona at UC Santa Barbara, the X-ray crystal structures of the enzyme, with and without the bound cysteine, have been solved. These structures reveal an active-site zinc ion that recognizes the cysteine thiol by forming the strong and specific zinc-thiolate bond. The zinc is located in a highly differentiated zinc center, composed of conserved amino acids that are important for catalysis of aminoacylation. The catalytic function of this zinc center, distinct from any other aminoacyl-tRNA synthetases, is being investigated by transient kinetics. This study will provide insight into one of ther most challenging issues of synthetases: the ability to alter specificity in order to create novel enzymes for expansion of the genetic code.
4. Aminoacylation in extreme environments.
Life is ubiquitous. The discovery of the archaea, the third branch of life, has identified life in extreme environments, including high temperatures (hyper thermophiles), high pressures (barophiles), high salinity (extreme halophiles) and anaerobes. This raises the question of how the archaea organisms adapt to extremes. Specifically, how do the archaea maintain their decoding specificity, with regards to tRNA structure, folding, and the tRNA-synthetase interactions that in mesophiles are extremely sensitive to temperature or salt. Because the archaea are important to the basal working of our planet and they account for the majority of our bio-mass, their biology, particularly the adaptation to extremes, is an essential issue. Also, because the extreme environments do not change rapidly, the archaea are considered more primitive than bacteria and eukarya and are thought to contain remnants of the origin of life. Intriguing features of tRNAs, aminoacyl-tRNA synthetases, and proteins that function as tRNA chaperons in extreme environments are being investigated.
5. tRNA and synthetases in diseases.
Because aminoacylation is essential to our cellular life, tRNAs and synthetases have both been targeted to develop new therapies for diseases and antibacterial reagents. In the case of CysRS, we have found that bacterial aminoacylation is dependent on the tRNA tertiary core, whereas the eukaryotic aminoacylation is independent of it. This provides a basis to study the tertiary core of the tRNA to identify agents that can target the bacterial aminoacylation. Second, tRNA genes have been found to be the hotspots for insertion of bacterial pathogenicity islands, which function like transposable elements that are essential for horizontal transfer of genes and diversification of bacterial chromosomes. We have raised the hypothesis that the tRNA genes associated with bacterial pathogenicity islands may encode the tRNA molecules necessary to stabilize the genetic transfer. This is an interesting possibility that will shed new light on the importance of tRNA beyond the well-recognized role in translation and replication. Third, tRNA molecules may be modified as a therapy to treat human diseases. One example is the neuro-degenerative Huntington's disease, where the triplet (CAG) expansion results in massive repeats of glutamine in the huntington protein, leading to toxic protein precipitation in the motor neurons. Based on our understanding of tRNA aminoacylation, we are creating modified tRNA molecules that may prevent the toxic protein precipitation.
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