Use of bgaH as a reporter gene for studying translation initiation in the archaeon Haloferax volcanii

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Sullivan, Eric L., S.M. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Uttam L. RajBhandary.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/43232

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/43232