| dc.description.abstracteng | Minimal cells are used to model and understand life’s complex processes. The most essential processes of life are thought to include cell replication, energy metabolism and genome maintenance. However, construction of a synthetic genome that entailed these core processes failed to yield a viable cell. This represents a gap in our knowledge regarding the functions required for life to unfold. To determine all the core processes required for life, naturally occurring minimal organisms that have undergone degenerative evolution, like Mycoplasma pneumoniae, are studied. Electron microscopy has shown that RNA polymerase and the ribosome form a super complex in some organisms. However, this coupling mechanism was not clear. In M. pneumoniae, NusA, an essential transcription factor with a disordered C-terminal region that contained crosslinks to the ribosome was investigated for this function. Truncated mutants of NusA that lacked this region were unable to grow, thus confirming the essentiality of this domain. NusA, rather than the proposed NusG, was thus proven to link the transcription and translation processes. Another approach to increase our understanding of life’s core processes is to consider organisms with already small genomes such as M. mycoides which have been synthetically minimized to possess only genes essential for growth in rich media. The resultant strain Syn3A, retains only half the original genome and contains 438 proteins. Given the essentiality criteria, it was a surprise that 149 genes had no specific function and a further subset of 79 genes were completely unannotated. As gene deletions are challenging in such a minimal organism, crosslinking experiments to elucidate protein-protein and protein-RNA interactions were performed. Along with confirming several known protein interactions, 28 uncharacterized proteins were found to interact with other proteins and 80 uncharacterized proteins have self-links. Four complexes containing uncharacterized proteins have been identified for further study and efforts to characterize 3 unknown proteins, Syn3A_0439, 0440 and 0505, were initiated. Self-links were also used to validate a predicted structure of 0439. Further, the complete subset of RNA-interacting protein machinery was thought to be retained in Syn3A. A second crosslinking project found 161 RNA-binding proteins that included 122 known RNA-binding proteins, 19 previously annotated proteins with a new RNA-binding function and 20 uncharacterized proteins. 4 of these 19 proteins and 6 of the 20 uncharacterized proteins were confirmed for RNA-binding ability. All the new proteins hold the possibility of undiscovered RNA-binding motifs, mechanisms and regulatory events. Specifically, as Syn3A_0317, 0388, 0439 and 0451 are all uncharacterized RNA-binding proteins that interact with other proteins, this work provides a direction for future investigations to follow. | de |