1. Abstract
Nucleosomes control transcription by blocking
transcription factors and RNA polymerase from
binding underlying DNA sequences. The goal of the
lab is to find out how nucleosomes position and
structure are modulated to regulate transcription.
Spt4 and Spt5 are conserved eukaryotic proteins
that form a protein complex, which associates with
elongating RNA polymerase II and controls proteins
that remove and reassemble nucleosomes over
transcribed genes. The C-terminal domain of Spt5
contains multiple repeats of the sequence
ST/AWGGA/Q, which are targeted by regulatory
kinases and act to recruit regulators of chromatin
structure. The hypothesis to be tested is that this C-
terminal region of Spt5 forms prion-like complexes.
Prions are proteins that convert between two
configurations, one of which is infectious. Prions in
this transmissible configuration are self-templating,
which allows them to convert other proteins into
the infectious configuration by mere contact. A
disease associated with the transmissible
configuration of prions is mad cow disease. To test
the hypothesis, full-length Spt5 and the C-terminus
of Spt5 will be fused to green fluorescent
protein. Fluorescent microscopy will be used to
monitor the ability of these proteins to form
aggregates. Kinase and phosphorylation site
mutants will be used to determine if Spt5’s
phosphorylation state affects its ability to
aggregate.
Methodology and Materials
• Spt5 Protein sequence and C-terminus fused
with green fluorescent protein
• Fluorescent microscopy used for analysis
• Spt5’s ability to phosphorylate analyzed for
affects to aggregate
Acknowledgments
Principal Investigator: Prof. Grant Hartzog
Research Supervisor: Michael Doody
Community College Liaison: Dr. Yves Tan
ACCESS Program: Professor Phil Crews, Director
Pamela D’Arcey, Associate Director
Steven Loveridge, Program Assistant
National Institutes of Health NIGMS Bridges to the future Program
(GM 51765-14)
Analysis and Conclusions
Gel analysis of our LR recombination that would
combine our gene of interest, Spt5 and C-terminus
sequence, to our fluorescent proteins did not give
us the results needed to verify that recombination
of those two vectors took place.
Through fluorescent microscopy, Spt5 protein
sequence and C-terminus part which contains
repeats of a sequence that recruit regulators of
chromatin structure, aggregates forms signifying
traces of prion-like complexes in C-terminus part
of Spt5 would have been seen. Figure 5. shows a
picture of what those clusters would have looked
like.
Pavel Morales, Grant Hartzog
University of California, Santa Cruz
Department of Molecular and Cellular Development
Literature and Resources cited
Hartzog, G.A., Fu J. 2012. The Spt4–Spt5 complex: A
multi-faceted regulator of transcription elongation.
Alberti S., Halfmann R. A Systematic Survey Identifies
Prions and Illuminates Sequence Features of
Prionogenic Proteins. Cell 137, 146-158. April 3, 2009.
Liu C., Change C., Chem Y. Spt4 is Selectively Required for
Transcription of Extended Trinucleotide Repeats. Cell
148, 690-701. February 17, 2012.
Figure 1 - "American Society for Microbiology Molecular
and Cellular Biology." The Spt5 C-Terminal Region
Recruits Yeast 3′ RNA Cleavage Factor I. N.p., n.d.
Web
Figure 2 – “Nucleosome.” Nucleosome. N.p., n.d. Web.
Figure 3 - "Green Fluorescent Protein." Wikipedia.
Wikimedia Foundation, 08 Oct. 2014. Web.
Future Work
Fluorescent Microscopy will be used to test the
C-terminal region of Spt5 for forms of prion-like
complexes.
I sure wish I’d
presented my
theory with a
poster before I
wrote my book.
Does transcription elongation factor Spt5 form prion-like complexes?
Background
Figure 1. Spt4-Spt5 Protein sits atop RNA Polymerase
II enzyme
Figure 3. Structure
of Green
Fluorescent Protein,
the reporter of
expression
Amplified Spt5-Cterminus
sequence using PCR
Created entry clones using
our PCR products with
pDONR221 as our donor
vector (BP recombination)
Inserted our entry vectors
into our destination vectors
using LR recombination
(Sup35, EGFP, EYFP)
Monitored Spt5-Cterminus
proteins to form aggregates
under the microscope
Digested with
restriction enzymes
to verify
recombination
Figure 4. Gel analysis
of digest of LR
products using AgeI
restriction enzyme .
Bands show two
fragments one at
around 700bp and the
other at 8000 bp.
Results
Prion:
• Protein that configures into a transmissible state
• Form aggregates
• Self-templating
Spt5:
• Removes nucleosomes from the path of RNA Polymerase II
 Reduces the frequency of transcription pausing
Nucleosomes:
• DNA in complex called chromatin
• Basic repeating unit of a chromatin
• 160 base pairs
Background Methodology and Materials
Figure 2. 160
base pairs
wrapped
around eight
histone protein
cores which
compose a
nucleosome
Figure 5. Arrows point to aggregates in the yeast cytosol