GUIDE RNA-DEPENDENT AND INDEPENDENT tRNA MODIFICATIONS IN ARCHAEA

Stable RNAs undergo a wide variety of post-transcriptional modifications, that add to the functional repertoire of these molecules. Some of these modifications are catalyzed by stand-alone protein enzymes, while some others are catalyzed by RNA-protein complexes. tRNAs from all domains of life contain many such modifications, that increase their structural stability and refine their decoding properties. Certain regions of tRNAs are more frequently modified than others. Two such regions are the anticodon loop, and the T&psi;C stem. In the halophilic euryarchaeon <italic>Haloferax volcanii</italic>, tRNA<super>Trp</super> and tRNA<super>Met</super>, both of which are transcribed as intron-containing pre-tRNA forms, contain Cm34 and &psi;54, in addition to other modifications, in these two regions, respectively. The Cm34 modification in both cases is RNP-mediated: tRNA<super>Trp</super> Cm34 formation being guided by its own intron, while that of tRNA<super>Met</super> being guided by a unique guide RNA called sR-tMet. We created genomic deletion of <italic>H. volcanii</italic> tRNA<super>Trp</super> intron by homologous recombination based technique, and showed that this strain is viable, and does not demonstrate any observable growth phenotype. However, the corresponding modifications are absent in this intron-deleted strain. Our structural and functional characterizations of sR-tMet revealed that it is unique in its structural properties and deviates considerably from its homologs in other Archaea. We also identified a novel L7Ae (a core protein associated with archaeal methylation guide RNPs) binding motif in sR-tMet. &psi;54, the near universal modification found in T&psi;C stem-loop of archaeal tRNAs is catalyzed by the protein Pus10. An earlier study from our laboratory had shown that Pus10 from two different archaea, <italic>Methanocaldococcus jannaschii</italic> (MjPus10) and <italic>Pyrococcus furiosus</italic> (PfuPus10) have differential activities towards &psi;54 formation. Using the crystal structure of Human Pus10 as template, we created homology models of MjPus10 and PfuPus10 proteins and identified several residues and motifs that might lead to this difference in activity. By a combination of both in vitro and in vivo mutational approaches, we confirmed several previously unidentified residues/motifs that serve as positive determinants of tRNA &psi;54 formation. Finally, as an extension to this study, we have identified a novel tRNA &psi;54 forming activity in mammalian nuclear extracts, and attributed this activity to Pus10.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Campus Only Dissertation

Discipline thesis:degree_discipline

Molecular Biology, Microbiology and Biochemistry

Year

2012

Author dc:creator

• Joardar, Archi

Contributors dc:contributor

• Gupta, Ramesh

• 2'-O-methylation
• Pseudouridine synthase
• tRNA modification

Repository record dc:identifier

https://opensiuc.lib.siu.edu/dissertations/625

OAI identifier oai:identifier

oai:opensiuc.lib.siu.edu:dissertations-1626