A genetic code expansion: investigation of UGA stop codon redefinition in selenoproteins

• Main Author: Baclaocos, Janinah
• Other Authors: Atkins, John F., Mackrill, John
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: University College Cork 2019
• Subjects: mRNA; Selenoprotein; Selenoprotein P; Selenium; Recoding; Ribosome; Translation; Genetic code; Frozen accident; Mollusc; Metazoa; Evolution; Ribosome profiling; Protein; Pacific; Crassostrea gigas; Pacific oyster
• Online Access: http://hdl.handle.net/10468/8558

After the genetic code was deciphered in the 1960s, Francis Crick formulated the ‘frozen accident’ hypothesis (Crick, 1968) to describe the origins of the genetic code as universal and resistant to change or evolution. Co-incidentally, evidence of the dynamic nature of genetic decoding emerged through a series of experimental observations which presented various cases of exceptions from what were known as the standard rules of decoding. There is now prevalent understanding and evidence that the genetic code is constantly evolving, and it can be altered by various organisms with possible implications for entire genomes or specific mRNAs. The incorporation of the 21st amino-acid selenocysteine in selenoproteins in response to the UGA translation ‘terminator’ codon is an example of a gene-specific expansion of the code. This thesis will deal primarily with two unique cases of UGA recoding. The first case is the synthesis of selenophosphate synthetase 1 (SPS1) (Chapter 2) whereby an unknown amino acid is inserted in response to a UGA codon in the hymenopteran honeybee, Apis mellifera, which lacks the machinery for Sec incorporation. The various attempts to characterize the amino acid inserted at this position by novel methods are described. In Chapter 3, the first extensive evolutionary analysis of the selenium transporting protein, selenoprotein P (SELENOP) in invertebrates is described with focused characterization in the mollusc, Pacific oyster, Crassostrea gigas. This unique case presented an unprecedentedly high Sec content (46 Sec) in the C-terminal domain of its SELENOP highlighting an extreme case of deviation from the standard genetic code read-out. It was shown that a supplemented heterologous system, was able to facilitate translation of oyster SelenoP mRNA up to the third or fourth Sec codon position of the distal region but was inadequate to produce the full-length protein. Further, the Sec-dedicated protein factor, the oyster SECIS binding protein 2 (SBP2) was characterized and its potential tested for ...