Objective
Two class II aminoacyl-tRNA synthetases will be studied, namely phenylalanyl-tRNA synthetase (PheRS) which can charge only tRNAPhe and alanyl-tRNA synthetase (AlaRS) which can charge both tRNAAla and 10Sa RNA (recognized by the ribosome as mRNA and tRNA simultaneously). It is of special interest to compare the structures of the enzymes that differ in the oligomeric structure and the tRNA identity set in order to reveal the regularities providing the specificity of tRNA (RNA) recognition.
High-resolution X-ray analysis will be used to study in detail the mechanism of tRNAPhe recognition with T. thermophilus PheRS, the most complicated enzyme of the aminoacyl-tRNA synthetases family. The crystallography of the PheRS-tRNA complexes obtained in the absence or in the presence of the two other substrates (ATP and phenylalanine) should reveal conformational differences of the protein and tRNA moieties in complexes corresponding to different steps of aminoacylation reaction. These data will be compared, extended and complemented with the data collection obtained in solution. tRNAPhe nucleotides of U- and G-containing regions, that are in close contact with PheRS, will be determined by using photoaffinity cross-linking. Functional investigation of the aminoacylation activity of the PheRS-tRNA complexes cross-linked at the definite positions would allow us to discriminate the contacts important for the initial tRNA binding from those involved in rearrangement of the complex during the catalytic step of the aminoacylation reaction.
The second line of crystallization experiments deals with AlaRS complexed with tRNAAla or 10Sa RNA. This data set as well will be backed up with detailed structural analysis performed in solution. The data obtained by the combination of photoaffinity cross-linking and foot printing techniques would allow us to determine precisely the nucleotide bases of 10Sa RNA involved into the interaction with AlaRS, to follow the conformational changes in RNA during the enzyme functioning. The comparison of these data with the similar data set for tRNAAla would allow us to understand the reason for multisubunit organization of the enzyme, whether such organization is necessary for the basic charging function or mainly for recognition of two different RNA molecules, which contain the same identity element at the acceptor arm of tRNA or tRNA-like structure. The analysis of the exact amino acid of the protein involved in the formation of the cross-links during the enzyme functioning is necessary for understanding transient structure of the complexes of tRNA and 10Sa RNA with the enzyme. Determination of the contacts of 10Sa RNA in the ribosome after binding and several steps of translocation and comparison of 10Sa RNA structure in the complexes with the ribosome and with AlaRS would allow us to detect specific protein-binding domains in 10Sa RNA and to suggest the structural model of 10Sa RNA in different complexes with proteins, thus to follow the structural rearrangement of the molecule during its functioning in the cell.
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Coordinator
76100 Rehovot
Israel
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