Amber Codon translation as pyrrolysine in methanosarcina Spp
The genes encoding the methylamine methyltransferases in Methanosarcina spp. each have an in-frame amber stop codon that is translated as pyrrolysine. This dissertation deals with various aspects of how this genetic encoding takes place. PylS is an aminoacyl-tRNA synthetase and Chapter 2 demonstrates that it is in fact a pyrrolysyl-tRNA synthetase. The pylS gene was cloned and its protein product was purified. In vivo experiments have subsequently shown that PylS has the ability to directly charge its cognate tRNA, tRNAPyl, with free pyrrolysine. The PylS/tRNAPyl pairing is the first example of a naturally occurring aminoacyl-tRNA synthetase/tRNA pair that can insert a non-canonical amino acid into protein. In Chapter 3 and the Appendix, in vivo and in vitro experiments were developed in order to further investigate UAG readthrough. In Chapter 3, an in vivo approach was developed in M. acetivorans in order to test whether a 3' downstream cis-acting element, termed the PYLIS element, is required for UAG translation. The PYLIS element was deleted from mtmB1 and this mutant gene was introduced into M. acetivorans. This strain, along with other strains bearing directed mutations of mtmB1, still produced MtmB1 that contained pyrrolysine. Although the amounts of MtmB1 production were lower than those seen in a strain bearing wild-type plasmid-based mtmB1, the lower amounts indicate that the PYLIS element may enhance, but is not essential for, UAG translation. This is the first experimental evidence that the PYLIS element is not required for amber codon readthrough in M. acetivorans. To determine whether any cis-acting elements are absolutely required for UAG translation, the E. coli uidA gene was employed as a reporter of UAG translation. Translation of uidA containing a UAG substitution gave dramatically different results, with UAG serving as a stop codon as well as a sense codon. The expression of UAG within a foreign gene indicates relaxed context for UAG translation. These results, along with the results from the PYLIS element deletion study, indicate that there is a high background of UAG readthrough in M. acetivorans, with a further enhancement of UAG translation in the presence of the PYLIS element.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:pyrrolysine trna methyltransferase methanosarcina pylis element pyls
Date of Publication:01/01/2006