Translational Regulation of Bovine Casein
Messenger RNA transcripts of αs2- and к-casein are translated at 25% of the efficiency of αs1- and β-casein transcripts; however, the molecular mechanisms governing the difference are unknown. We hypothesized that the bovine casein translational efficiency is influenced by characteristics of the untranslated regions (UTRs) and coding regions. The main objective of this study was to identify molecular mechanisms that explain differential translational regulation between bovine β- and αs2-casein by assessing the role of each putative translational regulatory factor found throughout full-length sequences in both in cellular and cell-free translation systems. This dissertation begins with the cloning and initial characterization of bovine β- and αs2-casein. Transcript analysis indicates that the two genes share similar characteristics of nucleotide sequence around the coding region and secondary structure. It is confirmed that αs2-casein mRNA has a lower translational efficiency compared to that of β-casein in a cell-free system. The latter portion of this thesis investigates further the UTRs and codon usage effect on difference in translational efficiency between β- and αs2-casein. Overall, our data suggest that β-casein 3’ UTR and αs2-casein 5’ UTR exert stimulatory effects on translation yet their effectiveness depends on the upstream and downstream sequences with which they are associated. Replacement of the UTRs of αs2-casein mRNA with those of β-casein did not stimulate translation. A stronger effect on translational efficiency was found in the coding region of αs2-casein which displays unfavourable codons at the 3’ terminus. Deletion of a 28-codon fragment from the 3’ terminus of the αs2-casein coding region increased translation to a par with β-casein. We suggest that the last 28 codons of αs2-casein is the main regulatory sequence that attenuates its expression and is responsible for the different translational expression of β- and αs2-casein mRNAs. Identification of regulatory factors that are responsible for translation efficiency improves our understanding of the molecular mechanisms of control of milk protein prodiction in secretory cells of the bovine mammary glands.