NSF Award
9904956
Hou<br/>9904956<br/>1. technical<br/><br/>The sequencing of the genome of Methanococcus jannaschii (M. jannaschii) is a necessary start to understand this archaeal organism, which is a strict anaerobic hyperthermophile that produces methane by using hydrogen to reduce carbon dioxide. The understanding of M. jannaschii will provide the basis to address its pathway of methane- production and to shed light on its basal machinery of processing genetic information and to relate this machinery to those of eubacteria and eucarya. However, while the genome of M. jannaschii encodes a full complement of tRNAs for decoding genetic information, it encodes only 16 of the 20 aminoacyl-tRNA synthetases that are responsible for attaching an amino acid to each tRNA. Missing in the genome are enzymes for glutamine, asparagine, lysine and cysteine. The absence of the glutamine and asparagine enzymes is explainable because the attachment of glutamine and asparagine to their tRNAs can be achieved by an alternative pathway. The absence of the lysine enzyme has been resolved by biochemical studies, which have elucidated an enzyme that catalyzes aminoacylation of tRNA with lysine using a motif distinct from that of the conventional lysine-tRNA synthetase. Unresolved at the moment is the enzyme for cysteine. The goal of this study is to use biochemical studies to address the missing enzyme in order to provide a better description of the archaeal genome. The specific aims are: 1. Investigate the hypothesis that the genome of M. jannaschii encodes a cysteine-tRNA synthetase that catalyzes aminoacylation of tRNA with cysteine. Test the hypothesis by purifying the enzyme to homogeneity and by cloning the gene for the enzyme and determination of the sequence of the enzyme. This will provide insight into the origin of the enzyme, its relationship with other cysteine-tRNA synthetases, and possible errors in the genomic sequence analysis. Characterize the enzyme for its mechanism of recognition of tRNA and determine if it discriminates against serine by an editing mechanism as the basis for an adaptation to extreme environments. 2. Investigate the hypothesis that the M. jannaschii cysteine-tRNA synthetase recognizes tRNA modifications for aminoacylation. Test the hypothesis by isolating fragments from the native tRNA that contain the important modifications to determine if it complements aminoacylation of the T7 transcript of the tRNA. Elucidate the identities of the important modifications and determine whether they include modifications of G37, G34, or G15. Nucleotides at position.<br/><br/><br/>2. non-technical<br/><br/>Life on this planet is structured in three primary domains, the eucarya, the eubacteria, and the archaea. Among these three, the archaea domain is the least understood. It consists of organisms from diverse and extreme habitats, which account for a large fraction of the microbial world. Many archaeal organisms have the ability to reproduce themselves from simple molecules, such as hydrogen, carbon dioxide, ammonia, sulfate, and sulfur. As such, they are essential for the basal workings of our biosphere. The sequencing of the genome of Methanococcus jannaschii (M. jannaschii) has opened the door to understanding the archaeal domain. This organism is a strict anaerobic hyper-thermophile that produces methane by using hydrogen to reduce carbon dioxide. The understanding of M. jannaschii will provide a basis to address its mechanism of methane-production and to gain insights into its ability to process genetic information from DNA to RNA to protein under extreme environmental conditions. Of particular interest is that while this genome encodes a full complement of transfers-RNAs ( tRNAs) forcarrying out protein synthesis, it encodes only a subset of the 20 enzymes thought to be required for tRNA functions. The goal of this project is to investigate the basis for some of the missing enzymes that are not identified from genomic analysis of M. jannaschii. Preliminary studies indicated evidence of these enzymes and raised the hypothesis that these enzymes may contain novel sequences such that they escape deletion by sequence analysis. Biochemical designs and strategies will be used to test the hypothesis and to enhance our understanding of this genome by complementing the genomic sequence analysis.
