1. KSU Symposium of Student Scholars 2015
REPSA-Directed Identification of DNA-Binding Specificity for Orphan Transcription Factors
Kamir Hiam
Van Dyke Lab
Dept. of Biochemistry
KSU Symposium of Student Scholars 2015

2. The Power of Modern Genetics
Genetic Sequencing  Raw Data
Series of bases ≠ cellular function
Further experimentation &
data analysis needed
Central Dogma of Biology
DNARNAProtein
Proteins responsible for the majority of cellular activities
Transcription Factors are proteins that bind to DNA to regulate expression

3. The Incomplete Tale of E. coli
Complete genome sequenced in 1997 (4.6+ Mbp)
4290 Open Reading Frames
240 potential TFs
Detailed binding profiles for only 68
Understanding orphan regulatory proteins can improve public health
Microbial disease
Human microbiome

4. REPSA and Combinatorial Methods
All combinatorial methods use large pools of randomized oligonucleotides difference in selection
(A) CASTing involves physical separation of bound protein-DNA complex requires knowledge of protein
(B) REPSA involves protein protection of enzymatic cleavage no prior knowledge of protein needed
REPSA optimal technique for transcription factor discovery

5. A Closer Look at REPSA Selection
Type IIS Restriction Enzymes cleave DNA at a defined distance from their recognition sites
Selection Template (68 bp)
5’ - CTAGGAATTCGTGCAGAGGTGAAT NNNNNNNNNNNNNNNNNNNN TTA CATCC CTCCAG AAGCTTGGAC – 3’ 3’ - GATCCTTAAGCACGTCTCCACTTA NNNNNNNNNNNNNNNNNNNN AAT GTAGG GAGGTC TTCGAACCTG – 5’
BsgI
HphI
FokI
BpmI

6. Orphan TF Specificity in the Van Dyke Lab
General Experimental Procedure
Clone bacteria (E. coli K12) with gene of interest
Induce protein expression
Perform combinatorial selection for sequence specificity (REPSA)
Sequence final pool of DNA
Analyze data for consensus sequence
Verify, Hypothesize, & Publish
Repeat!

7. LexA as model protein for REPSA
Previously characterized repressor protein
SOS response regulator
Known consensus binding sequence
DNase I protection
EMSA
Weight Matrix Studies
Combinatorial methods never used
Ideal candidate for REPSA protocol optimization
Overall structure of E. coli LexA-DNA complex

8. Representative Round of REPSA
Protein/
No Enzyme
No Protein/
Enzyme
6 Rounds
PCR
9 Rounds
12 Rounds

9. Unexpected Consensus Sequences
Round 7: 553/1000 sequences e=4.0e-290
What are structural features of T-rich sequences?
Why would this inhibit cleavage?
Round 14: 923/1000 sequences e=9.3e-2093
Doesn’t this sequence look familiar?
Why would this inhibit cleavage?
According to previous literature, FokI cleaves DNA without regard for sequence specificity… Yet we appear to have found violations of this rule.

10. Future Direction LexA Specificity Studies
Work out issues with protein binding
Examine Orphan Transcription Factors
In E. coli and other organisms such as extremophiles
Mixtures of Transcription Factors
Develop REPSA techniques to handle multiple TFs at one time
Ultimate goal to work with less pure cellular extracts

11. Acknowledgements
Van Dyke Lab Members NSF STEM Scholarship LSAMP Scholarship OVPR KSU Foundation NIH