The ribosome is a 2.5 MDa molecular machine that converts the information encoded in mRNA into protein, following the rules defined by the genetic code. In all organisms protein composition is limited to 20 amino acids, with the rare exceptions of pyrrolysine and selenocysteine. Although rarely used in the proteome, the incorporation of selenocysteine into proteins is essential for life in many organisms, including humans. The mRNA encoding a selenoprotein has a stem loop, known as a selenocysteine insertion sequence (SECIS) element, following a UGA stop codon that facilitates the ribosome to introduce selenocysteine at the stop codon. This requires a unique set of factors used only for the synthesis and insertion of selenocysteine including a dedicated tRNA, translation factor and enzymes that enable the conversion of serine to selenocysteine. The human proteome includes 25 selenoproteins that are mostly uncharacterised because of the inability to express them in bacteria. This is due to the divergence of RNA and protein factors as well as the inherently low efficiency of selenocysteine incorporation in bacteria. I have developed a reporter gene that provides an efficient life/death selection for selenocysteine incorporation. Using directed evolution, a library of random 16S rRNA mutants was screened for clones with an altered ability to incorporate selenocysteine. Six point mutations were identified in the 16S rRNA which affect selenocysteine incorporation efficiency, one conferring a four-fold increase in efficiency without affecting canonical translation. This result was validated by measuring the reduction of benzyl viologen in an endogenous context with the E. coli selenoprotein formate dehydrogenase H.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2012|