1. Does transcription elongation factor
Spt5 form prion-like complexes?
Pavel Morales
Hartnell College
ACCESS Summer Research Institute 2014
Grant Hartzog
University of California, Santa Cruz
Department of Molecular and Cellular Development

2. Prion
Protein that converts between two configuration, one of
which is infectious.
Prions in this transmissible/infectious configuration are
self-templating.
Form aggregates (clusters)
Associated with Mad Cow Disease and Creutzfeldt-Jakob
Disease

3. Spt5 Protein
Plays an important role in transcription elongation in the
nucleus of the cell.
Binds elongating RNA polymerase II
Reduces the frequency of transcription pausing.
Recruits chromatin regulators and RNA processing factors to
elongation complexes

4. Nucleosomes
Eukaryotes store their DNA
in the nucleus as a protein-
DNA complex called
chromatin.
Basic repeating unit of a
chromatin
160 basepairs of DNA
The spatial arrangement of
nucleosomes on a gene,
influences its transcription.

5. S T/A W G G A/Q Sequences
The C-terminus domain of Spt5 contains multiple repeats of this sequence.
C-terminus part of Spt5, predicted to form a prion-like domain.
The are targeted by regulatory kinases and act to recruit regulators of
chromatin structure.
1 2 3 4 5 6
931 S S W G G A
937 S T W G G A
948 S A W G G A
958 S A W G G Q
969 S T W G G A
975 S A W G N K
981 S S W G G A
987 S T W A S G
1000 S T W G G T
1009 *S A Y G G A
1015 *S T W G G N
1032 *S A W G N Q
1043 *S A W N N Q
1052 S N Y G G N
1058 S T W G G H
T A
Consensus S A W G G Q
No. of matches 15 12 13 13 11 9
SequencePosition
Six amino-acid repeat at the c-terminus of Spt5
* Sites of
phosphorylation for
Spt5 HP

6. Goal of my Research/Hypothesis
C-terminal region of Spt5 forms prion-like
complexes

7. Methodology
 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.

8. Amplified Spt5-Cterminus sequence
from wild-type and mutant yeast
strains 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 if recombination
took place
Detailed Methodology

9.  3k bp mark
 1k bp mark
8k bp mark
DNA
Digest
Destination
Vector
2,2a EYFP
2,2b EYFP
2,3a EGFP
2,3b EGFP
2,3c EGFP
2,3d EGFP
2,3e EGFP
3,2a EYFP
3.3a EGFP
3,3b EGFP
Expected fragments:
~8000 bp
~750 bp

10. Predicted Results/Conclusions
 Alberti S., Halfmann R. A Systematic Survey Identifies Prions and Illuminates Sequence Features
of Prionogenic Proteins. Cell 137, 146-158. April 3, 2009.

11. Future Work
 Monitor the ability of these proteins using fluorescent
microscopy for aggregates

12. Acknowledgements
Principal Investigator: Grant Hartzog
Research Supervisor: Michael Doody
Community College Liaison: 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)