Prokaryotic Gene Regulation: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Chromosome Conformation Capture in Bacteria and Archaea
	1 Introduction
	2 Materials
		2.1 Cell Fixation
		2.2 3C Library Preparation
		2.3 3C-qPCR
		2.4 3C-Seq
	3 Methods
		3.1 Cell Fixation
			3.1.1 Escherichia coli
			3.1.2 Saccharolobus solfataricus
		3.2 3C Library Preparation
			3.2.1 Lysis and Solubilization: Escherichia coli
			3.2.2 Lysis and Solubilization: Saccharolobus solfataricus
			3.2.3 Restriction Digestion
			3.2.4 Fractionation
			3.2.5 Ligation
			3.2.6 Reverse Cross-Linking and DNA Purification
		3.3 3C-qPCR
			3.3.1 Experimental Design
			3.3.2 Experimentation
			3.3.3 Data Analysis
		3.4 3C-Seq
			3.4.1 NGS Library Preparation
			3.4.2 Data Analysis and Generation of Contact Maps
	4 Notes
	References
Chapter 2: Micrococcal Nuclease Digestion Assays for the Analysis of Chromosome Structure in Archaea
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Chromosome Extraction
		2.3 MNase Digestion
		2.4 Agarose Gel Electrophoresis
	3 Methods
		3.1 Cell Culture
		3.2 Chromosome Extraction
		3.3 MNase Digestion
		3.4 Agarose Gel Electrophoresis
		3.5 Interpretation of the Results
	4 Notes
	References
Chapter 3: Detecting DNA Methylations in the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius Using SMRT Sequenci...
	1 Introduction
	2 Materials
		2.1 Sulfolobus acidocaldarius DSM639 Culture
		2.2 Genomic DNA Extraction
		2.3 (Optional Step) Preparation of the Negative Control by Performing a Whole-Genome Amplification (WGA)
		2.4 Genomic DNA Quality Check
		2.5 PacBio Single-Molecule Real-Time (SMRT) Sequencing and Analysis
	3 Methods
		3.1 Sulfolobus acidocaldarius DSM639 Culture
		3.2 Genomic DNA Extraction from Sulfolobus acidocaldarius Cells
		3.3 (Optional Step) Preparation of the Negative Control by Performing a Whole-Genome Amplification (WGA)
		3.4 DNA Quality Check (See Note 12)
		3.5 PacBio SMRT Sequencing and Analysis
			3.5.1 Sequencing (See Note 14)
			3.5.2 Analysis (See Fig. 1)
	4 Notes
	References
Chapter 4: ProD: A Tool for Predictive Design of Tailored Promoters in Escherichia coli
	1 Introduction
	2 Materials
		2.1 Relevant Repository Files
		2.2 Prerequisites If ProD Is Run from the Terminal
		2.3 Prerequisites If ProD Is Utilized Using Jupyter
	3 Methods
		3.1 The ProD Tool
		3.2 Construction of Promoters with Desired TIF
			3.2.1 Using the Terminal
			3.2.2 Using Jupyter
			3.2.3 Cloning Design
		3.3 Predicting TIF by Evaluation of Custom Spacers
			3.3.1 Using the Terminal
			3.3.2 Using Jupyter
	4 Notes
	References
Chapter 5: Computational Study on the Dynamics of Mycobacterium Tuberculosis RNA Polymerase Assembly
	1 Introduction
	2 Materials
		2.1 Protein Data Bank (PDB)
		2.2 PyMOL
		2.3 Modeller
		2.4 TM-Align
		2.5 The FoldX Suite
		2.6 NACCESS
		2.7 Protein Interactions Calculator (PIC)
		2.8 Programming Tools
			2.8.1 ProDy
			2.8.2 Biopython
			2.8.3 Bio3d
	3 Methods
		3.1 Dataset Preparation
		3.2 Structural Features
			3.2.1 Structural Similarity
			3.2.2 Analysis of Protein-Protein Interface
		3.3 Features of Dynamics
			3.3.1 Square Fluctuations
			3.3.2 Dynamic Cross-Correlation Matrix
			3.3.3 Perturbation Response Scanning (PRS)
	4 Conclusion
	5 Notes
	References
Chapter 6: In Vitro Transcription Assay for Archaea Belonging to Sulfolobales
	1 Introduction
	2 Materials
		2.1 Safety Instructions
		2.2 Purification of Transcription Factors
			2.2.1 Solutions, Enzymes, Chemicals
			2.2.2 Other Materials and Equipment
		2.3 Purification of RNA Polymerase
			2.3.1 Solutions, Enzymes, Chemicals
			2.3.2 Other Materials and Equipment
		2.4 Preparation of [5′-32P] Single-End Labeled Oligonucleotides and DNA Fragments
			2.4.1 Solutions, Enzymes, Chemicals
			2.4.2 Other Materials and Equipment
		2.5 Native Gel Electrophoresis for Purification of Oligonucleotides
			2.5.1 Solutions and Chemicals
			2.5.2 Other Materials and Equipment
		2.6 Autoradiography
			2.6.1 Solutions
			2.6.2 Other Materials and Equipment
		2.7 Recovery of DNA from Polyacrylamide Gel
			2.7.1 Solutions, Chemicals, Materials, and Equipment
		2.8 In Vitro Transcription Reactions
			2.8.1 Solutions, Chemicals, Materials, and Equipment
		2.9 Reverse Transcription Reactions
			2.9.1 Solutions, Chemicals, Materials, and Equipment
		2.10 Denaturing Gel Electrophoresis
			2.10.1 Solutions and Chemicals
			2.10.2 Other Materials and Equipment
	3 Methods
		3.1 Purification of Transcription Factors
			3.1.1 Transformation of Plasmids into Overexpression Strain
			3.1.2 Heterologous Expression (in E. coli) and Purification of S. solfataricus TBP and TFB
		3.2 Expression and Purification of S. solfataricus RNA Polymerase
		3.3 Preparation of [5′-32P] Single-End Labeled Primer
			3.3.1 Primer Characteristics
			3.3.2 Oligonucleotide Labeling
		3.4 Purification of [32P]-Labeled DNA by Non-denaturing Polyacrylamide Gel Electrophoresis
			3.4.1 Aim
			3.4.2 Treatment of Glass Plates
			3.4.3 Preparation of a 10% Non-denaturing Polyacrylamide Gel
			3.4.4 Sample Preparation and Gel Electrophoresis
			3.4.5 Imaging by Autoradiography
			3.4.6 Recovery of Labeled Primer from the Polyacrylamide Gel Matrix
		3.5 In Vitro Transcription (IVT)
			3.5.1 Design and Preparation of Template DNA
			3.5.2 In Vitro Transcription Reactions
		3.6 Reverse Transcription
		3.7 Analysis of the Reaction Products by Gel Electrophoresis Under Denaturing Conditions
			3.7.1 Preparation of an 8% Denaturing Acrylamide/bis-acrylamide Stock Solution
			3.7.2 Treatment and Assembly of the Glass Plates
			3.7.3 Gel Preparation
			3.7.4 Sample Preparation, Gel Electrophoresis Under Denaturing Conditions, and Visualization by Autoradiography
		3.8 Densitometry and Interpretation
	4 Notes
	References
Chapter 7: Prediction of DNA-Binding Transcription Factors in Bacteria and Archaea Genomes
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Retrieving Genome Sequences
		2.3 Retrieving a Repertoire of DNA-Binding TFs with Experimental Evidence
		2.4 PFAM Assignments to Predict  TFs
		2.5 Identification of Orthologous Proteins
		2.6 BLAST Searches
		2.7 HMMER Searches
	3 Methods
		3.1 PFAM Assignments in Sequenced Genomes
		3.2 Identification of Protein Orthologs
		3.3 Integration of Outputs to TF Assignments
		3.4 Prediction of Helix Turn Helix (HTH)
		3.5 Prediction of TFs Based on Deep Learning Methods
	4 Discussion
	References
Chapter 8: In Vivo Screening Method for the Identification and Characterization of Prokaryotic, Metabolite-Responsive Transcri...
	1 Introduction
	2 Materials
		2.1 Bacterial Strains, Media, and Solutions
		2.2 Molecular Biology
		2.3 Plate Reader Measurements
	3 Methods
		3.1 Design and Construction of a Two-Plasmid Reporter System
			3.1.1 Promoter Reporter Construct (pPRC)
			3.1.2 Inducible TF Construct (pITC)
			3.1.3 Preparation of Cell Extract for Assembly Cloning
			3.1.4 Construction of pPRC and pITC by Polymerase Chain Reaction and Assembly Cloning
		3.2 Development and Screening of the Transcription Factor Library
			3.2.1 Selection of TFs and Corresponding Promoter Sequences
			3.2.2 Construction of Reporter Strains
			3.2.3 Fluorescence Measurements
		3.3 Modifications to Tune Reporter Output and Allow Further Characterization
			3.3.1 Adaptation of a Heterologous Promoter to Improve Transcription in E. coli
			3.3.2 Changing the TF/Promoter Ratio by Transitioning to a Single-Plasmid System
			3.3.3 Elimination of Crosstalk with Native Metabolism by Using E. coli Knock-out Mutants
	4 Notes
	References
Chapter 9: Application of Special Electron Microscopy Techniques to the Study of DNA - Protein Complexes in E. coli Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Dormant E. coli Cells BL21-Gold (DE3)/pET-DPS
		2.2 Equipment Used for Preparation of Bacterial Culture
		2.3 Specimen Preparation for Electron Microscopy
		2.4 Equipment for Specimen Preparation for Electron Microscopy
		2.5 Equipment Used for Electron Microscopy and Electron Tomography
		2.6 Software Used for Electron Microscopy and Electron Tomography
	3 Methods
		3.1 Bacterial Culture Preparation
			3.1.1 Preparation of a Bacterial Inoculum
			3.1.2 Bacteria Cultivation
			3.1.3 Storage of Bacterial Cultures
		3.2 Specimen Preparation for Electron Microscopic Studies
			3.2.1 Washing Procedure
			3.2.2 Preparation for Transmission Electron Microscopy and Tomography Experiments
			3.2.3 Preparation for EDS
			3.2.4 Thin-Section Carbon Coating
		3.3 Transmission Electron Microscopy and Electron Tomography
			3.3.1 Single-Axis Tomography Data Acquisition
			3.3.2 Tomography Data Processing
			3.3.3 Fourier-Filtering the Crystal Lattice TEM Images
		3.4 Energy Dispersive Spectroscopy (EDS)
	4 Notes
	References
Chapter 10: High-Speed Atomic Force Microscopy Visualization of Protein-DNA Interactions Using DNA Origami Frames
	1 Introduction
	2 Materials
		2.1 DNA Origami Design and Synthesis
		2.2 Protein Solution
		2.3 DNA Origami AFM Sample Preparation and AFM Imaging
	3 Methods
		3.1 DNA Origami Design
		3.2 Construction of the 74-mer Duplex DNA Fragments
		3.3 DNA Origami Synthesis
		3.4 Sample Preparation for AFM Imaging
		3.5 Pre-imaging Setup for High-Speed AFM of Protein-DNA Interactions in fluid
		3.6 High-Speed AFM Imaging of Protein-DNA Interactions in fluid
			3.6.1 Imaging of the DNA Origami Frames
			3.6.2 Imaging of Protein - DNA Interactions
			3.6.3 Image Analysis
	4 Notes
	References
Chapter 11: Separation and Characterization of Protein-DNA Complexes by EMSA and In-Gel Footprinting
	1 Introduction
	2 Materials
		2.1 Safety Instructions
		2.2 Preparation of [5′-32P] Single-End Labeled DNA Fragments
			2.2.1 Solutions, Enzymes, Chemicals
			2.2.2 Other Materials and Equipment
		2.3 Native Gel Electrophoresis
			2.3.1 Solutions and Chemicals
			2.3.2 Other Materials and Equipment
		2.4 Autoradiography
			2.4.1 Solutions
			2.4.2 Other Materials and Equipment
		2.5 Recovery of DNA from Polyacrylamide Gel
			2.5.1 Solutions and Chemicals
			2.5.2 Other Materials and Equipment
		2.6 EMSA
			2.6.1 Solutions and Products
			2.6.2 Other Materials and Equipment
		2.7 In-Gel Footprinting
			2.7.1 Solutions and Chemicals
			2.7.2 Other Materials and Equipment
		2.8 Preparation of A+G and C+T Sequencing Ladders
			2.8.1 Solutions and Chemicals
			2.8.2 Other Materials and Equipment
		2.9 Denaturing Gel Electrophoresis
			2.9.1 Solutions and Chemicals
			2.9.2 Other Materials and Equipment
	3 Methods
		3.1 Preparation of [5′-32P] Single-end Labeled DNA Probes by PCR Amplification
			3.1.1 DNA Probe Characteristics
			3.1.2 Oligonucleotide Labeling and PCR Amplification
		3.2 Purification of [32P]-Labeled DNA by Nondenaturing Polyacrylamide Gel Electrophoresis
			3.2.1 Aim
			3.2.2 Treatment of Glass Plates
			3.2.3 Preparation of a 6% Nondenaturing Polyacrylamide Gel (see Note 6)
			3.2.4 Sample Preparation and Gel Electrophoresis
			3.2.5 Imaging by Autoradiography
			3.2.6 Recovery of Labeled DNA from the Polyacrylamide Gel Matrix
		3.3 Optimization of Protein-DNA Complex Formation by EMSA
		3.4 In-Gel Footprinting (see Fig. 2 for a Schematic Outline and Example)
		3.5 Preparation of Chemical Sequencing Ladders (Maxam and Gilbert Reactions)
			3.5.1 A+G Sequencing Ladder
			3.5.2 C+T Sequencing Ladder
		3.6 Analysis of the Reaction Products by Gel Electrophoresis Under Denaturing Conditions
			3.6.1 Prepare a Denaturing Acrylamide/Bis-Acrylamide Stock Solution of Suited Concentration.
			3.6.2 Treatment and Assembly of the Glass Plates
			3.6.3 Gel Preparation
			3.6.4 Sample Preparation, Gel Electrophoresis Under Denaturing Conditions and Visualization by Autoradiography
	4 Notes
	References
Chapter 12: Chemical Protection and Premodification-Binding Interference for the Identification of Phosphate and Base-Specific...
	1 Introduction
		1.1 Purine Methylation
		1.2 Depurination and Depyrimidation
		1.3 Thymine Oxidation
		1.4 Cytosine Modification with Hydroxylamine Hydrochloride
		1.5 Uracil Substitution
		1.6 Phosphate Ethylation
	2 Materials
		2.1 Safety Measures
		2.2 Preparation of [5′-32P] Single-End Labeled DNA Fragments
			2.2.1 Solutions, Enzymes, Chemicals
			2.2.2 Other Materials and Equipment
		2.3 Native Polyacrylamide Gel Electrophoresis for DNA Purification and EMSA
			2.3.1 Solutions and Chemicals
			2.3.2 Other Materials and Equipment
		2.4 Autoradiography
			2.4.1 Solutions
			2.4.2 Other Materials and Infrastructure
		2.5 Recovery of DNA from Polyacrylamide Gel
			2.5.1 Solutions and Chemicals
			2.5.2 Other Materials and Equipment
		2.6 Denaturing Polyacrylamide Gel Electrophoresis
			2.6.1 Solutions and Chemicals
			2.6.2 Other Materials and Equipment
		2.7 Methylation Protection
			2.7.1 Products and Solutions
			2.7.2 Other Materials and Equipment
		2.8 Chemical Modification Reactions for Premodification-Binding Interference Assays
			2.8.1 Products and Solutions Common to All DNA Modification Reactions
			2.8.2 Methylation of Purines with DMS
			2.8.3 Depurination at Low pH and High Temperature
			2.8.4 Depyrimidation with Hydrazine
			2.8.5 Thymine Oxidation with KMnO4
			2.8.6 Cytosine Modification with NH2OH.HCl
			2.8.7 Uracil Substitution
			2.8.8 Phosphate Ethylation with N-Ethyl-Nitrosourea
		2.9 EMSA
			2.9.1 Solutions and Products
			2.9.2 Other Materials and Equipment
	3 Methods
		3.1 Synthesis of [5′-32P] Single-End Labeled DNA (see Note 4)
		3.2 Purification of [32P]-labeled DNA
			3.2.1 Aim
			3.2.2 Treatment of Glass Plates
			3.2.3 Preparation of a 6% Nondenaturing Polyacrylamide Gel
			3.2.4 Sample Preparation and Gel Electrophoresis
			3.2.5 Imaging by Autoradiography
			3.2.6 Recovery of Labeled DNA from the Polyacrylamide Gel Matrix
		3.3 Methylation Protection
			3.3.1 Protein Binding and DNA Methylation
			3.3.2 Strand Cleavage at Methylated Gs Only
			3.3.3 Strand Cleavage at Methylated Gs and As
			3.3.4 Sample Preparation for Gel Electrophoresis Under Denaturing Conditions
			3.3.5 Preparation of a Denaturing Acrylamide/Bis-Acrylamide Stock Solution of Suited Concentration
			3.3.6 Preparation of a Denaturing Polyacrylamide Gel (see Note 15)
			3.3.7 Gel Electrophoresis Under Denaturing Conditions and Visualization by Autoradiography
		3.4 Premodification-Binding Interference Assays
			3.4.1 Preparation of Sparingly Modified DNA
			3.4.2 Depurination
			3.4.3 Depyrimidation
			3.4.4 Thymine-Specific Oxidation with KMnO4
			3.4.5 Cytosine-Specific Modification with NH2OH.HCl
			3.4.6 Uracil Substitution
			3.4.7 Phosphate Ethylation with N-Ethyl-Nitrosourea
		3.5 EMSAs with Sparingly Modified DNA
		3.6 Cleavage of Sparingly Modified DNA After Recovery from Gel
			3.6.1 Reactions Common to Several Modifications
			3.6.2 Strand Scission of Methylated DNA
			3.6.3 Strand Scission at Ethylated Phosphates
			3.6.4 Strand Scission of Uracil-Substituted DNA
		3.7 Separation of the Reaction Products by Gel Electrophoresis Under Denaturing Conditions
		3.8 Potential Additional Investigations
	4 Notes
	References
Chapter 13: Analysis of Protein-DNA Interactions Using Isothermal Titration Calorimetry: Successes and Failures
	1 Introduction
	2 Materials
		2.1 Preparation of Duplex DNA
		2.2 Proteins
		2.3 Buffers
	3 Methods
		3.1 Sample Dialysis
		3.2 Preparation of Solutions for the ITC Experiment
		3.3 Performing an ITC Experiment
		3.4 Analysis of Results
			3.4.1 Integration of Thermograms
			3.4.2 Analysis of Binding Isotherms
		3.5 Advanced Experiments
			3.5.1 Temperature-Dependent Experiments
			3.5.2 Experiments at Different Ionic Strengths
			3.5.3 Nonbinary Reactions
		3.6 Interpretation of Thermodynamics Parameters
		3.7 Unexpected Results
			3.7.1 Aggregation
			3.7.2 Air Bubbles
			3.7.3 No Signal
			3.7.4 High Dilution Effects
			3.7.5 Unexpected Stoichiometry
			3.7.6 Affinity Is Too Low
			3.7.7 Affinity Is Too High
	4 Notes
	References
Chapter 14: Determination of RNA Structure with In Vitro SHAPE Experiments
	1 Introduction
	2 Materials
		2.1 Precautionary Measures
			2.1.1 Working RNase-Free
			2.1.2 Working with Ethidium Bromide
			2.1.3 Working with Radioisotope 32P
		2.2 Preparing the in vitro Transcription Template
		2.3 T7 In Vitro Transcription
		2.4 RNA Purification
		2.5 Validation of RNA Integrity with RNA Gel Electrophoresis
		2.6 RNA Folding and RNA Modification with the NMIA SHAPE Reagent
		2.7 Primer Extension
			2.7.1 [γ-32P] Labeling of the Reverse Transcription Primer
			2.7.2 Reverse Transcription of the Modified RNA
			2.7.3 Preparing the Dideoxy Chain-Termination Sequencing Ladder
		2.8 Denaturing Acrylamide Gel Electrophoresis
			2.8.1 Preparing the Denaturing Acrylamide Gel
			2.8.2 Acrylamide Gel Electrophoresis
		2.9 Autoradiography and Interpretation and Quantification of the SHAPE Results
	3 Methods
		3.1 Designing the DNA Template and Reverse Transcription Primer for In Vitro Transcription
		3.2 Preparing the In Vitro Transcription Template
		3.3 T7 in vitro Transcription
		3.4 RNA Purification
		3.5 Validation of RNA Integrity with RNA Gel Electrophoresis
		3.6 RNA Folding and RNA Modification with the NMIA SHAPE Reagent
		3.7 Primer Extension
			3.7.1 [γ-32P] Labeling of the Reverse Transcription Primer
			3.7.2 Reverse Transcription of the Modified RNA
			3.7.3 Preparing the Dideoxy Chain-Termination Sequencing Ladder
		3.8 Denaturing Acrylamide Gel Electrophoresis
			3.8.1 Preparing the Denaturing Acrylamide Gel
			3.8.2 Acrylamide Gel Electrophoresis
		3.9 Autoradiography
		3.10 Interpretation and Quantification of the SHAPE Results
	4 Notes
	References
Chapter 15: Microscale Thermophoresis to Study RNA-RNA Binding Affinity
	1 Introduction
	2 Materials
		2.1 Interaction Partners
		2.2 Buffers and Reagents
		2.3 Instruments and Software
		2.4 Materials
	3 Methods
		3.1 In Vitro Transcription of  RNA
		3.2 PCI Clean-up to Remove the DNase from Generated  RNA
		3.3 3′ End Labeling of Generated  RNA
		3.4 Plan the Experiment
		3.5 Run the Experiment
		3.6 Analyze the  Data
	4 Notes
	References
Chapter 16: Toeprint Assays for Detecting RNA Structure and Protein-RNA Interactions
	1 Introduction
	2 Materials
		2.1 DNA Template for In Vitro Transcription (see Note 2)
		2.2 RNA Template Preparation (see Note 1)
		2.3 5′ End-Labeled Primer
		2.4 Toeprint Reaction
		2.5 Sequencing  Gel
	3 Methods
		3.1 DNA Template Containing a T7 RNAP Promoter
		3.2 RNA Preparation and Purification
		3.3 Preparation of 5′ End-Labeled Primer
		3.4 Sequencing Ladder
		3.5 Toeprint Reaction with E. coli CsrA and ymdA RNA to Demonstrate How Protein-Binding Can Alter RNA Structure
		3.6 30S Ribosomal Toeprint with E. coli ymdA RNA to Demonstrate How CsrA Can Affect Ribosome Binding
		3.7 Toeprint Analysis of Tylosin-induced Ribosomal Stalling in the tlrB Leader Using PURExpress
	4 Notes
	References
Index