This thesis describes work done with the thyroid hormone receptor (TR), a nuclear
protein which binds to specific DNA sequences and regulates transcription in response to
thyroid hormone levels. The studies can be divided into two broad categories:
structure/function studies of the TR protein, particularly with regards to DNA binding
function; and, structure/function studies of the DNA sequences to which the thyroid
hormone receptor binds in order to regulate gene transcription.
In order to examine the DNA binding properties of the TR an electrophoretic mobility
shift assay (EMSA) was utilized. Conditions of this assay were optimized for the use of in
vitro translated TR. Mutant forms of the β-isoform of thyroid hormone receptor were
generated using a PCR-based mutagenesis protocol. Each mutant substituted a different
residue of the 12 amino acid-long α-recognition helix with alanine. The mutants were
analyzed for their abilities to bind to thyroid hormone response elements (TREs), and to
activate transcription in transfected eukaryotic cells. The DNA binding results were
consistent with a conserved α-helix structure, with conserved function for many residues,
that is similar to that of the related receptors for glucocorticoids and estrogen. Only the first
of the three non-conserved residues lying in the P-box (EGG), a portion of the recognition
α-helix that facilitate differential binding of distinct DNA sequences, disrupted binding
when substituted with alanine. The third position of the P-box, when substituted with
alanine exhibits an altered ability to bind to certain natural TREs. The mutant form of TR
with alanine substituted for the second P-box position displayed only a modest decrease in
DNA binding affinity compared to wild-type TR (roughly 3-fold), yet was completely
deficient in trans-activation.
The structure-function studies of TR binding sites on DNA applied a methylation
interference protocol to examine the interactions of TR with direct repeats (DR) of the
idealized hexameric sequence, spaced by three to five base-pairs. The interactions of both
the TR/TR homodimer and the TR/RXR (9-cis-retinoic acid receptor) heterodimer with the
DRs were examined. The methylation interference patterns for the TR/TR homodimer
bound to the DR sequences are virtually identical for spacers of four and five base-pairs,
but with three base-pairs, there is some evidence that at least one DNA binding domain is
misaligned with the DNA to accomodate the unfavourable spacer length. The TR/RXR
heterodimer methylation interference pattern is distinct on all three DRs, probably due to the
fact that in the heterodimer cooperative intermolecular contacts are made between the DNA
binding domains of the two receptors, but only when the spacer distance is four base-pairs.
When a poorly conserved everted repeat (EvR) that overlaps the idealized DR is present.
the homodimer, but not the heterodimer, binds this cryptic EvR in competition with the
DR. The binding modality of the TR homodimer and TR/RXR heterodimer to DRs was
reevalutated using point mutants and EMSA. The TR homodimer and TR/RXR
heterodimer both bind to idealized direct repeats with DBDs aligned appropriately for a
direct repeat; however, evidence is presented that there are certain poorly conserved
sequences that are intermediate between DRs and EvRs that are differentially recognized by
the TR homodimer and the TR/RXR heterodimer. That is, the homodimer binds with the
DBDs aligned appropriately for a EvR, and the heterodimer DBDs are aligned appropriately
for a DR. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9701 |
| Date | 13 July 2018 |
| Creators | Faris, Jonathan Scott |
| Contributors | Romaniuk, Paul John |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
Page generated in 0.0022 seconds