Eukaryotic ribosome biogenesis, a dynamic and coordinated multistep process which requires more than 150 trans-acting factors, has been intensely studied in the yeast Saccharomyces cerevisiae. This evolutionarily conserved process involves numerous cleavages of pre-rRNA, modification of nucleotides, and concomitant assembly of the ribosomal proteins onto the rRNA. Considerable information is available about the importance of conserved pre-rRNA cleavage events in ribosome biogenesis; however, very little is known about the exact role of modified nucleotides, which cluster within the functionally important regions of the ribosome. One conserved group of modifications is the dimethylation of two adjacent adenosines at the 3´ end of the small subunit rRNA which is ubiquitously carried out by the Dim1/KsgA methyltransferase family. Although dimethylation and KsgA are dispensable for survival in bacteria, the eukaryotic enzyme Dim1 is essential because of its requirement in the early pre-rRNA processing events. Similarly, few other members of the family have also evolved to carryout a second unrelated function in the cell. Almost all of the information about Dim1 was obtained from in vivo experiments in yeast, and has been determined that it is an indispensable part of a RNA-protein complex carrying out the pre-rRNA processing. Sequence analysis clearly shows that eukaryotic and archaeal enzymes have an extra insert in their C-terminal domain which is absent in bacterial enzymes and a better understanding of Dim1's function is only possible by its structural characterization which is the aim of this study. After several attempts, the yeast Dim1p was expressed under mild conditions in E. coli and purified in soluble form. Dim1p was able to methylate bacterial 30S subunits both in vivo and in vitro, indicating its ability to recognize bacterial substrate. Supporting our hypothesis, neither the bacterial nor archaeal orthologs were able to complement the processing function of Dim1p in yeast, tested using the plasmid shuffling technique. Our results suggest that the C-terminal insert of Dim1p, along with some structural features of the N-terminal domain, is important for its function in pre-rRNA processing. Further studies are required to understand the complex interactions between proteins and RNA involved in the ribosome biogenesis.

The dioxin receptor (DR), a transcription factor with basic-helix-loop-helix/PERARNTSIM (bHLH/PAS) homology domains, is activated by toxic xenobiotic ligands leading to severe physiological disturbances most of which are due to deregulation of receptor’s central role in normal development. Activation mechanisms of DR in the presence of exogenous or endogenous ligands are poorly understood. Elucidation of factors involved in the activation of the receptor would assist not only in development of an optimal measure for risk assessment of levels of common environmental pollutants but also in providing novel targets for therapeutic interventions. Posttranslational modifications (PTMs) play an indispensable role in all major signal transduction pathways by increasing the inventory of chemical modifications beyond those already present in the side-chains of common amino acids. Thus, by simple on/off or complex patterns generated by these PTMs, they control a myriad of different biological outcomes. Numerous studies that have suggested an important role of posttranslational modifications in DR activation has prompted a search in this direction, however, apart from phosphorylations at Ser36 and Ser68 no other PTM sites are known. Advanced mass spectrometry (MS)-based characterisation of PTMs is an established technique that can comprehensively provide an accurate cast of all PTM variants and their locations on a protein. This thesis reports the first MS-based comprehensive characterisation of all PTM sites of the purified latent DR and preliminary analysis of identified PTM sites of the activated DR in response to developmental signals (suspension-activated DR) and signals leading to toxic outcomes (ligand-activated DR). The PTM map of the latent DR revealed from this study comprises of 25 phosphorylations, 4 monomethyl-lysines, 2 dimethyl-lysines, 1 O-acetyl-serine and 2 O-sulfono-serines. Most of the phosphorylations and other PTMs were present in the conserved regions of the protein. Investigation of the activated samples of the receptor revealed loss of the above repertoire of modifications and possible presence of some rarer modifications such as O-acetyl-serines in suspension-activated instead of O-sulfonations and pyrophosphorylation at Ser716 in both suspension- as well as ligand-activated DR. A comprehensive mutagenesis study is in progress to understand the functional consequence of each of these modification sites and unravel the functional posttranslational system in DR signalling.

The dioxin receptor (DR), a transcription factor with basic-helix-loop-helix/PERARNTSIM (bHLH/PAS) homology domains, is activated by toxic xenobiotic ligands leading to severe physiological disturbances most of which are due to deregulation of receptor’s central role in normal development. Activation mechanisms of DR in the presence of exogenous or endogenous ligands are poorly understood. Elucidation of factors involved in the activation of the receptor would assist not only in development of an optimal measure for risk assessment of levels of common environmental pollutants but also in providing novel targets for therapeutic interventions. Posttranslational modifications (PTMs) play an indispensable role in all major signal transduction pathways by increasing the inventory of chemical modifications beyond those already present in the side-chains of common amino acids. Thus, by simple on/off or complex patterns generated by these PTMs, they control a myriad of different biological outcomes. Numerous studies that have suggested an important role of posttranslational modifications in DR activation has prompted a search in this direction, however, apart from phosphorylations at Ser36 and Ser68 no other PTM sites are known. Advanced mass spectrometry (MS)-based characterisation of PTMs is an established technique that can comprehensively provide an accurate cast of all PTM variants and their locations on a protein. This thesis reports the first MS-based comprehensive characterisation of all PTM sites of the purified latent DR and preliminary analysis of identified PTM sites of the activated DR in response to developmental signals (suspension-activated DR) and signals leading to toxic outcomes (ligand-activated DR). The PTM map of the latent DR revealed from this study comprises of 25 phosphorylations, 4 monomethyl-lysines, 2 dimethyl-lysines, 1 O-acetyl-serine and 2 O-sulfono-serines. Most of the phosphorylations and other PTMs were present in the conserved regions of the protein. Investigation of the activated samples of the receptor revealed loss of the above repertoire of modifications and possible presence of some rarer modifications such as O-acetyl-serines in suspension-activated instead of O-sulfonations and pyrophosphorylation at Ser716 in both suspension- as well as ligand-activated DR. A comprehensive mutagenesis study is in progress to understand the functional consequence of each of these modification sites and unravel the functional posttranslational system in DR signalling.