Bacterial Regulatory RNA, 1st ed. 2018 Methods and Protocols Methods in Molecular Biology Series, Vol. 1737

This volume details the most important methods used for studying prokaryotic non-coding RNAs and their protein accomplices. Chapters present methods in sections covering different aspects of the biology of that field: identification of ncRNAs, their differential expression, characterization of their structure, abundance, intracellular location and function, their interaction with RNA binding proteins, and plausible applications of ncRNA elements in the rapidly emerging field of synthetic biology. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Bacterial Regulatory RNA: Methods and Protocols serves as a guidebook for scientists working toward the development of new tools and procedures for the vital field of sRNA biology.

Part I: Identification of sRNA

1. Workflow for a Computational Analysis of an sRNA Candidate in Bacteria

Patrick R. Wright and Jens Georg

2. Molecular Phylogenomics for the Identification and Characterization of trans-encoded Small Non-coding RNA Families

Antonio Lagares (Jr) and Claudio Valverde

3. Bioinformatic Approach for Prediction of CsrA/RsmA-regulating small RNAs in Bacteria

Carl T. Fakhry, Kourosh Zarringhalam, and Rahul V. Kulkarni

Part II: Function and Differential Expression of sRNA

4. Host-pathogen Transcriptomics by Dual RNA-seq

Alexander J. Westermann and Jörg Vogel

5. Identification of New Bacterial Small RNA Targets using MS2 Affinity Purification Coupled to RNA Sequencing

Marie-Claude Carrier, Guillaume Laliberté, and Eric Massé

6. Assessment of External Guide Sequences’ (EGS) Efficiency as Inducers of RNase P-mediated Cleavage of mRNA Target Molecules

Saumya Jani, Alexis Jackson, Carol Davies-Sala, Kevin Chiem, Alfonso Soler-Bistué, Angeles Zorreguieta, and Marcelo E. Tolmasky

7. Evaluating the Effect of Small RNAs and Associated Chaperones on Rho-dependent Termination of Transcription in vitro

Cédric Nadiras, Annie Schwartz, Mildred Delaleau, and Marc Boudvillain

8. A Method to Map Changes in Bacterial Surface Composition Induced by Regulatory RNAs

Pierre Fechter

9. Fluorescence-based Methods for Characterizing RNA interactions in vivo

A. N. Leistra M. K. Mihailovic and L. M. Contreras

10. Mutational Analysis of sRNA-mRNA Base Pairing by Electrophoretic Mobility Shift Assay

Eva Maria Sternkopf Lillebæk and Birgitte Haahr Kallipolitis

11. An Integrated Cell-free Assay to Study Translation Regulation by Small Bacterial Non-coding RNAs

Erich Michel, Olivier Duss, and Frédéric H.-T. Allain

Part III: Quantitation and Subcellular Localisation of sRNA

12. Quantitative Super-resolution Imaging of Small RNAs in Bacterial Cells

Seongjin Park, Magda Bujnowska, Eric McLean, and Jingyi Fei

13. Extraction and Analysis of RNA Isolated from Pure Bacteria—Derived outer Membrane Vesicles

Janine Habier, Patrick May, Anna-Heintz-Buschart, Anubrata Ghosal, Anke K. Wienecke-Baldacchino, Esther N.M. Nolte-'t Hoen, Paul Wilmes, and Joëlle V. Fritz

14. Absolute Regulatory Small Noncoding RNA Concentration and Decay Rates Measurements in Escherichia coli

Florent Busi, Véronique Arluison, and Philippe Régnier

Part IV: Cofactors of sRNA-based Regulation

15. High-resolution, High-throughput Analysis of Hfq Binding Sites using UV-crosslinking and Analysis of cDNA (CRAC)

16. Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly

Kimberly A. Stanek and Cameron Mura

17. Single Molecule FRET Assay to Observe the Activity of Proteins Involved in RNA/RNA Annealing

18. Techniques to Analyse sRNA-protein Cofactor Self-assembly in vitro

David Partouche, Antoine Malabirade, Thomas Bizien, Marisela Velez, Sylvain Trepout, Sergio Marco, Valeria Militello, Christophe Sandt, Frank Wien, and Véronique Arluison

19. Sequence-specific Affinity Chromatography of RNA-binding Proteins from Bacterial Cells

Jonathan Gans, Jonathan Osborne, Juliet Cheng, Louise Djapgne, and Amanda G. Oglesby-Sherrouse

20. Identification of Small RNA-protein Partners in Plant Symbiotic Bacteria

Marta Robledo, Ana M. Matia-González, Natalia I. García-Tomsig, and José I. Jiménez-Zurdo

Part V: Applications in Synthetic Biology

21. A Modular Genetic System for High-throughput Profiling and Engineering of Multi-target Small RNAs