Transcript Slide 1

Protein-protein interactions between widely-expressed and testis specific subunits of TFIIA and TFIID in
Drosophila melanogaster
Leah Hirschman, Mark Hiller
Department of Biological Sciences
Goucher College
Abstract
The regulation of transcription plays an important role in cellular
development and differentiation. Spermatogenesis is an excellent
model to explore the regulation of tissue-specific transcription. In
Drosophila melanogaster, General Transcription Factors TFIIA and
TFIID work together during transcription initiation. TFIID is a complex
composed of TATA-binding protein (TBP) and fourteen TATA binding
protein-associated factors (TAFs). TFIIA is comprised of three subunits,
including the TFIIAγ subunit. In the testes, there is a homolog of the
TFIIAγ subunit, which is encoded by the gene tfiia-s-2. Also in the
testes, there are additional testis-TAFs (tTAFs) which are components
of TFIID. Homologues of TBP, TBP-related factors 1 and 2 (TRF1 and
TRF2), are found in the testes and other tissues. With so many different
versions of proteins located in the testes, there are many possible
combinations of proteins that could aid in the regulation of transcription
in spermatogenesis. To study the possible interactions between widely
expressed proteins and testis specific proteins, GST pull-down assays
were used to analyze protein-protein interactions in vitro. By studying
the interactions between proteins that regulate transcription in
spermatogenesis, we will create a more comprehensive model of the
complexes that regulate testis-specific transcription.
TRF1
L H
1
2
TRF2
L H
3
4
C
5
45.0
TRF2
TRF1
31.0
21.5
Table 1: The General Transcription Factors TFIIA and TFIID are
essential for transcription initiation. TFIID is comprised of TBP and TBP-associated
factors (TAF’s). In many tissues, TBP homologues (TRF1 and TRF2) are expressed. Five TAF
homologues are expressed only in the testis. TFIIA is comprised of three subunits: α, β, and γ. The
testis-specific homologue TFIIAγ-2 is expressed in the testes and is encoded by the gene tfiia-s-2.
MW in kDa
Figure 4: TRF1 and TRF2 proteins were synthesized
in vitro using a transcription/translation system.
Recombinant V5 epitope tagged TRF1 and TRF2 proteins were expressed
in vitro and visualized by Immuno-blot using anti-V5 antibody. Lanes 1 and
2 are TRF1 while lanes 3 and 4 are TRF2. The high concentration lanes
contain 3.75 times more protein than the low concentration lanes. One lane
is used for the TRF V5-antibody control. A recombinant construct
expressing TBP is under construction.
Figure 2: Amino acid alignment of TFIIA γ-proteins. The generally expressed
Human (hTFIIAg) and fly (dTFIIA-S) TFIIA proteins are aligned along with testis-specific
homologues. Two spliced forms (dTFIIA-S-2a and dTFIIA-S-2b) are shown. Black shading indicates
identity while grey shading indicates similarity within the amino acid sequences. Sections α1 and α2
indicate α-helix secondary structure and sections β1-3 indicate β-sheet secondary structure. Testisspecific TFIIA-S-2b has extra amino acids in between sections α1 and α2 that are not seen in any
other version of TFIIA.
GST Pull Down
Figure 5: GST pull down assays can detect proteinprotein interactions between TBP/TAF homologues and
TFIIA. E. coli expressed TFIIA is tagged with glutathione s-transferase (GST).
GST tagged proteins will bind to glutathione-coated agarose beads and can be
purified. Any proteins that interact with the GST tagged proteins will also remain
bound to the beads and can be co-purified. Three forms of TFIIA are expressed in
E. coli, including the widely expressed form and two testis-specific forms. GST pull
down assays can detect interactions between more than two proteins.
Figure 1: Spermatogenesis is an excellent model
system to explore tissue-specific transcription
regulation. Spermatogenesis begins when a testis stem cell divides
and one subsequent daughter cell undergoes four cycles of mitosis. After
these mitotic divisions, high levels of transcription regulation occur.
Following this growth and gene expression phase, cells undergo meiotic
divisions and cellular differentiation to produce sperm cells.
Future Directions
Figure 3: Model of the crystal structure derived from the TFIIA/TBP/DNA
complex from yeast. TFIIA (red, blue, and green) and TATA-binding protein (TBP, a subunit of
TFIID, orange) are known to interact with each other and with DNA (yellow) during transcription initiation.
There is a possibility that testis-specific homologues of TFIIA (TFIIAγ-2), TBP homologues TRF1 and TRF2,
and testis-specific TAF’s (tTAF’s) are also involved in the regulation of transcription during
spermatogenesis.
• Continue to analyze protein-protein interactions in vitro
using GST pull down assays
• Create a recombinant construct expressing TBP
• Create a more comprehensive model of the proteinprotein complexes that aid in transcription regulation
Acknowledgments:
• Goucher College for funding