Graduation Semester and Year

2012

Language

English

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Advisor

Shawn Christensen

Abstract

Non-LTR retrotransposons encode a conserved cysteine histidine motif of undetermined function. The spacing of the cysteines and histidine residues in the motif is CX₃CX₇HX4C. The site specific non-LTR retrotransposon R2Bm inserts into a specific site within the 28S ribosomal gene. Two subunits of the R2 protein, bound to distinct segments of the element RNA, are involved in the integration reaction. The subunit bound to the 5′ protein binding motif of the element RNA binds to target DNA downstream of the insertion site. The subunit bound to the 3′ protein binding motif of the element RNA binds to target DNA upstream of the insertion site. The two segments of the element RNA cause RNP specific protein conformational changes to occur to their respectively bound R2Bm proteins. Of the four predicted nucleic acid binding domains encoded by the R2Bm protein--two RNA binding domains and two DNA binding domains--only downstream DNA binding activity has been mapped to a specific domain of the R2Bm protein. The role of the CCHC motif in nucleic acid binding, protein conformation, and target primed reverse transcription related enzymatic activities were probed using purified components and a C/SC/S double point mutant of the CCHC motif. The CCHC motif does not appear to be involved in direct recognition of target DNA as the net DNA binding capacity of the C/SC/SHC mutant was on par or better than WT R2Bm protein as was net DNA cleavage. The observed DNA cleavage pattern, however, was aberrant with little to no cleavage at the insertion site and promiscuous cleavage elsewhere. The mutant protein-DNA complexes showed mobilities that differed from WT in the presence of and absence of RNA. Most strikingly the C/SC/SHC mutant had a diminished protein conformational response to the presence of RNA. The few subunits that did form the correct conformation were apparently able to cleave the bottom DNA strand and perform target primed reverse transcription. We conclude that the CCHC motif modulates RNA associated protein conformation changes.

Disciplines

Biology | Life Sciences

Comments

Degree granted by The University of Texas at Arlington

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