Pyrrolysine is the 22nd genetically encoded amino acid found in nature. This dissertation examines processes central to the translation of UAG as pyrrolysine.
The pylT gene encoding the tRNA for pyrrolysine, tRNAPyl, is part of the pyl operon of Methanosarcina spp. which also contains the pylS gene, encoding an aminoacyl-tRNA synthetase, PylS; and the pylB, pylC, and pylD genes proposed to catalyze the synthesis of pyrrolysine. In the first analysis, the essentiality of pyrrolysine incorporation in methanogenesis from methylamines is tested by characterizing a Methanosarcina acetivorans mutant with the pylT gene region deleted. This study reveals the phenotype of a strain that can not decode UAG as pyrrolysine.
The data in the second study shows that PylS catalyzes the ATP-dependent activation of synthetic pyrrolysine. Taken together with results obtained by others the data indicates that PylS is a pyrrolysyl-tRNA synthetase capable of directly ligating pyrrolysine to tRNAPyl in vitro and in vivo. These results prove that PylS is the first aminoacyl-tRNA synthetase to be discovered from nature that is capable of attaching a genetically-encoded amino acid, not one of the common twenty, to cognate tRNA. Further, the data indicates that the pylB, pylC, and pylD gene products are sufficient for pyrrolysine synthesis from precursors common to M. acetivorans and E. coli.
Earlier studies indicated that the two canonical lysyl-tRNA synthetases in Methanosarcina spp. form a complex and attach lysine to tRNAPyl. In the third study, the relevance of this indirect route is tested in vivo. The data presented demonstrates that the route of tRNAPyl aminoacylation involving lysyl-tRNA synthetase complex formation is not required for UAG translation as pyrrolysine.
In the final study, the substrate specificity of PylS is probed using analogs of pyrrolysine. Features of the pyrroline ring of pyrrolysine that are determinants of PylS catalyzed activation are identified.