Use of bgaH as a reporter gene for studying translation initiation in the archaeon Haloferax volcanii
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259808091-MIT.pdf
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Author(s)
Sullivan, Eric L., S.M. Massachusetts Institute of Technology
Advisor(s)
Uttam L. RajBhandary.
Date Issued
2008
Publisher
Massachusetts Institute of Technology
Abstract
The bgaH gene isolated from Haloferax lucentensis codes for P-galactosidase. To study the function of initiator tRNAs in translation initiation in Haloferax volcanii, the initiator AUG codon of the bgaH gene was mutated to UAG, UAA, UGA, and GUC. Four different H. volcanii initiator tRNA derived mutants with complementary anticodons were also made. When plasmids carrying the bgaH reporter and mutant initiator tRNAs were coexpressed in H. volcanii, the UGA and GUC decoding tRNAs were aminoacylated, but functional 0-galactosidase was produced only in the presence of the latter tRNA. This result confirms that translation can initiate with some alternative codons, but suggests that the amino acid attached to the tRNA also plays a role. It is unknown if leaderless transcripts will have similar requirements, therefore mutant bgaH reporters lacking 5' untranslated regions were also generated. I also describe modifications of the bgaH reporter for studying suppression of termination codons in H. volcanii. The serine codon at position 184 of the bgaH gene was mutated to the termination codons UAA and UAG. H. volcanii serine tRNA derived suppressor tRNAs with complementary anticodons were also generated. These suppressor tRNAs should allow a study of the requirements for suppression of UAG and UAA codons in H. volcanii, in particular the question of whether suppressors of the UAA codon can also suppress the UAG codon in archaea. H. volcanii WFD 11 used as the host does not have any endogenous 3-galactosidase.
(cont.) I have shown that extracts made from H. volcanii transformants can be used to assay for 3-galactosidase using either O-Nitrophenyl-p-galactoside or Beta-Glo reagent as a substrate. This latter assay couples the D-Luciferin product of cleavage of 6-O-P-galactopyranosyl-luciferin by P-galactosidase to the more precise and sensitive luciferase assay. Since little is known about translation in archaea, future work will involve modifying identity elements in the initiator tRNA to study their requirements in both initiation and elongation in archaea.
Description
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.
Includes bibliographical references (leaves 30-32).
Subjects
Biology.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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