oral presentation; Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria. SIGNIFICANCE Organisms must constantly make regulatory decisions in response to a change in cellular state or environment. However, while the catalog of genomes expands rapidly, we remain ignorant about how the genes in these genomes are regulated. Here, we show how a massively parallel reporter assay, Sort-Seq, and information-theoretic modeling can be used to identify regulatory sequences. We then use chromatography and mass spectrometry to identify the regulatory proteins that bind these sequences. The approach results in quantitative base pair-resolution models of promoter mechanism and was shown in both well-characterized and unannotated promoters in Escherichia coli. Given the generality of the approach, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in a wide range of bacteria.

1. Systematic Approach For Dissecting
Molecular Mechanism of Transcription
Nathan M. Belliveau, Stephanie Barnes, William Ireland, Daniel Jones,
Michael Sweredoski, Annie Moradian, Sonja Hess, Justin Kinney, and Rob
Phillips.
MAY 22, 2018, 121(15)
Proceedings of the National Academy of Sciences of the United States of America
Babita Neupane
M.Sc. Bioinformatics, 2nd Batch
FRONTIERS IN MOLECULAR BIOLOGY | TRANSCRIPTION REGULATION IN PROKARYOTES | Dr. NAMRAJ DHAMI

2. Outline of Presentation
About PNAS
AboutAuthors
Before Getting Started
Introduction
General Overviewof Approach
Methodology
Results
Summary
Discussions
ResearchImpact

3. 2 0 X X C O N F E R E N C E P R E S E N T A T I O N 3
Proceedings of the National Academy of Sciences of the United States of America
(PNAS)
✓ Peer reviewed journal of National Academy of Sciences(NAS).
✓ Impact factor: 11.1
✓ Ranking: 8/73
✓ Acceptance rate:15.3%
✓ Multidisciplinary journal; most cited, comprehensive and publishes
more than 3,500 research papers annually.
✓ First journal Vol 1(1) - Jan 1915;latest journal published Vol 120(31).
✓ We are discussing paper published in Vol 121(15) under section of
Biophysics and Computationalbiology in PNAS.

4. About Authors

5. PROMOTORS
✓ Region of DNA where transcription of a gene is
initiated. Promoters are a vital component of
expression vectors because they control the
binding of RNA polymerase to DNA.
REGULATORYBINDING SITES IN
PROMOTORS
✓ Specific DNA sequences within or near gene
promoters where regulatory proteins (transcription
factors) bind and regulates gene expression.
✓ Transcription factors recognize and bind to these
binding sites to either enhance (activators) or
inhibit (repressors) transcriptionof target genes.
BEFORE GETTING STARTED

6. INTRODUCTION
✓ Sequencing Revolution
✓ RegulonDB and EcoCyc.
✓ Chromatin immunoprecipitation (ChIP).
✓ Massively parallel reporter assays; CRISPR array.
✓ Systematic Approach = Massively parallel reporter assays + Affinity
Chromatography + Mass Spectrometry + Information-theoretic modeling
✓ Systematic study of multiple bacterial promoters, extracting nucleotide-
resolution models in known as well as unknown bacteria.
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 6

7. General Overview of the Approach
✓Sort Seq : To identify binding sites in promotor; expression and energy Matrix
Model.
✓Affinity Chromatography: To isolate regulatory proteins (TFs) from binding sites.
✓Mass Spectrometry : To characterize regulatory protein based on abundance. Used
to calculate Protein Enrichment Ratio.
✓Sort Seq experiments in gene deletion strains; comparison with previous
expression shift and energy matrix model ; functional mechanism of each regulatory
proteins.
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 7

8. METHODOLOGY

9. 1. Sort- Seq
✓Used in molecular biology and
genomics to study the relationship
between genotype and gene
expression at the single-cell level.
✓Combines two methods:
fluorescence-activated cell sorting
(FACS) and high-throughput
sequencing.
✓In cell sorting, cells are labeled
with fluorescent markers or
reporters that indicate specific
genetic features or expression
levels.
✓The labeled cells are then sorted
using flow cytometry or a cell
sorter (e.g., Beckman Coulter
MoFlo XDP) into separate bins
based on the fluorescencesignals.
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 9
Sequence Logos
Energy Matrix Modeling
Expression Shift Analysis
Illumina Sequencing.
Fluorescence-Activated Cell Sorting (FACS)
Promotor Library Generation

10. FACS/EXPRESSION SHIFT/ ENERGY MATRICES
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 10
Expression Shift
Energy Matrices of Lacl binding site.
Florescence activated cell sorter(FACS)

11. 2. DNAAffinity Chromatography and Mass Spectrometry
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 11
Protein Enrichment Calculation
Mass Spectrometry Analysis
DNA Affinity Chromatography
Stable Isotopic Labeling of Cell Culture
(SILAC) Approach
Experimental & Mutated Oligonucleotide
Pool
Stable Isotope Labeling by Amino acids in Cell
culture(SILAC)
DNA Affinity Chromatography

12. 3. Collection and Preparation of Deletion Strain
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 12
Organism used was E. coli K-12.
Deletion strains were created using the Lambda red recombinase
method.
Deletion Strain collected from Yale University(lysA, purR, xylE)
and developed by researcher (lacZYA, relBE, marR, and dgoR).

13. 4. Sort Seq with deletion strain
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 13
Sort Seq performed to analyze Expression Shift and Energy
Matrices.
Comparison of Energy Shift and Energy Matrices to
validate functional role of regulatory proteins in gene
expression.

14. 4. Code availability and data analysis
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 14
✓ The code were used for processing data and plotting, as well as the final
processed data, plasmid sequences, and primer sequences.
✓ It can be found on the GitHub repository of the RPGroup-PBoC
(https://www.github.com/RPGroup-PBoC/sortseq belliveau; DOI:
10.5281/zenodo.1184169).

15. RESULTS

16. General Approach Used

17. A. RECOVERING THE REGULATORY FEATURES OF WELL
CHARACTERIZED PROMOTERS USING SORT-SEQ.
a. LacZYA(lac)
Responsible for lactose metabolism in E.coli and other bacteria.
lacZ-: β-galactosidase; cleave lactose into galactose and glucose
lacY: lac permease: lactose uptake
lacA: transacetylase: transfers an acetyl group from CoA to the hydroxyl group of
galactosides.
3 binding sites observed
Lac 01
CRP
Lac 03

18. Result

19. RECOVERING THE REGULATORY FEATURES OF WELL
CHARACTERIZED PROMOTERS USING SORT-SEQ.
b. relBE promotor (Bacterial toxin/antitoxin system)
✓ It transcribes the toxin–antitoxin pair RelE and RelB.
✓ Important roles in cell physiology, including cellular persistence .
✓ Toxin RelE excess --> cleaves mRNA --> Cellular paralysis.
✓ Antitoxin RelB excess --> repressorof its own promoter --> prevent transcription

20. Results
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 20

21. RECOVERING THE REGULATORY FEATURES OF WELL
CHARACTERIZED PROMOTERS USING SORT-SEQ.
b. marRAB
✓ Activates multidrug resistance efflux pump, ArcAB-TolC, and increases antibiotic
tolerance.
✓ Activated by own promotor marbox binding site.
✓ Transcription further enhanced by Fis.
✓ Repressed by marR.

22. Results
2 0 X X C o n f e r e n c e p r e s e n t a t i o n 22

23. B. SORT-SEQ DISCOVERS REGULATORY
ARCHITECTURES IN UNANNOTATED
REGULATORY REGIONS.

24. The purT promoter contains a simple
repression architecture and
is repressed by PurR.
a. purT Promotor
✓ Catalyze the third step in de novo purine
biosynthesis.
✓ In close association with yebG promotor.
✓ Contain binding sites for purT and RNAP.
✓ Validated performing sort seq in presence of
purine derivative hypoxanthine.
yebG Promotor
✓ Induced during DNA damage.
✓ Contain binding sites for LexA and RNAP.
✓ Validated with sort seq in presence of Mitomycin.

25. The xylE operon is induced in the
presence of xylose mediated
through binding of XylR and CRP.
b. xylE Promotor
✓ Involved in uptake of xyloses.
✓ RNAP binding site located between −80 and
−40 bp relative to the xylE gene.
✓ Might have multiple binding sites.
✓ Energy matrices suggest presence of CRP
binding sites
✓ Activation of RNAP might be mediated by
both CRP and XylR.

26. The dgoR promoter is autorepressed by DgoR with transcription
mediated by class II activation by CRP
c. dgoR Promotor
✓ This operon codes for D-galactonate–catabolizing enzymes.
✓ RNAP binding site between −30 and −70 bp relative to the native start codon for
dgoR

28. ✓ Sort-Seq successfully used to dissect the functional mechanisms of regulatory sequences
in bacteria.
✓ Involves using a reporter assay to identify functional transcription factor binding sites and
then enriching and identifying the TFs using DNA oligonucleotides and mass spectrometry
analysis.
✓ Energy matrices used to model and infer the DNA binding specificity of regulatory
factors.
✓ This was validated by examining previously annotated promoters and successfully
identifying regulatory mechanisms.
SUMMARY

29. ✓ Challenges include the need for quantitate protein abundance datasets and transcript
abundance datasets for a better understanding of regulatory mechanisms in bacteria.
✓ The approach can be applied to any promoter sequence and can be further enhanced by using
microarray-synthesized promoter libraries and landing pad technologies for chromosomal
integration.
✓ Techniques that combine these assays with transcription start site readout can provide
additional resolution in identifying regulatory binding sites.
✓ DNA affinity chromatography samples can be multiplexed using isobaric labeling strategies to
identify transcription factors across many target binding sites.
✓ Continued improvements in mass spectrometer sensitivity and sample processing will make
the assay less onerous to apply across many targets and different binding conditions.
✓ Reporter assays in transcription factor deletion strains and genetic perturbations using
CRISPR interference can provide secondary means of identifying and validating binding sites.
DISCUSSION

30. Thank you