Does transcription elongation factor Spt5 form prion-like complexes?

Pavel MoralesHartnell College

ACCESS Summer Research Institute 2014

Grant HartzogUniversity of California, Santa Cruz

Department of Molecular and Cellular Development

PrionProtein 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

Spt5 ProteinPlays an important role in transcription elongation in the

nucleus of the cell.Binds elongating RNA polymerase IIReduces the frequency of transcription pausing.Recruits chromatin regulators and RNA processing factors to

elongation complexes

NucleosomesEukaryotes store their DNA

in the nucleus as a protein-DNA complex called chromatin.

Basic repeating unit of a chromatin

160 basepairs of DNAThe spatial arrangement of

nucleosomes on a gene, influences its transcription.

S T/A W G G A/Q SequencesThe 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 SW G G A

937 S TW G G A

948 S AW G G A

958 S AW G G Q

969 S TW G G A

975 S AW G N K

981 S SW G G A

987 S TW A S G

1000 S TW G G T

1009 *S AY G G A

1015 *S TW G G N

1032 *S AW G N Q

1043 *S AW N N Q

1052 S NY G G N

1058 S TW G G H

TA

Consensus S A WG G Q

No. of matches 15 12 1313 11 9

SequencePosition

Six amino-acid repeat at the c-terminus of Spt5

* Sites of phosphorylation for Spt5 HP

Goal of my Research/Hypothesis

C-terminal region of Spt5 forms prion-like complexes

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.

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

1kb

3k bp mark

1k bp mark

8k bp mark2,2a

2,2b

2,3a

2,3b

2,3c

2,3d

2,3e

3,2a

3,3a

3,3b

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

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.

Future Work Monitor the ability of these proteins using fluorescent

microscopy for aggregates

Acknowledgements

Principal Investigator: Grant Hartzog Research Supervisor: Michael DoodyCommunity College Liaison: Yves Tan

ACCESS Program: Professor Phil Crews, DirectorPamela D’Arcey, Associate DirectorSteven Loveridge, Program Assistant

National Institutes of Health NIGMS Bridges to the future Program (GM 51765-14)