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Coordinated Regulation of Gene Expression by E.Coli Chromatin Proteins FIS and H-NS

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Coordinated Regulation of Gene Expression by E.Coli Chromatin Proteins FIS and H-NS Coordinated regulation of gene expression by E.coli chromatin proteins FIS and H-NS A Dissertation Presented by Ramesh Mavathur Nanjundaiah A thesis Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biochemistry School of Engineering and Science Jacobs University Bremen Coordinated regulation of gene expression by E.coli chromatin proteins FIS and H-NS by Ramesh Mavathur Nanjundaiah A thesis Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biochemistry Approved as to style and content by: _________________________________________ Prof. Dr.Georgi Muskhelishvili ___________________________________ Prof. Dr. Albert Jeltsch ___________________________________ Prof. Dr. Sebastian Springer ___________________________________ Prof. Dr. Andrew Travers Date of Defense: August 29th 2007 School of Engineering and Science Parts of this work has been published in: Auner, H., Buckle, M., Deufel, A., Kutateladze, T., Lazarus, L., Mavathur, R., Muskhelishvili, G., Pemberton, I., Schneider, R., and Travers, A. (2003) Mechanism of transcriptional activation by FIS: role of core promoter structure and DNA topology. J Mol Biol 331: 331-344. Blot, N., Mavathur, R., Geertz, M., Travers, A., and Muskhelishvili, G. (2006) Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome. EMBO Rep 7: 710-715. Lang, B., Madan Babu, M., Blot, N., Bouffartigues, E., Buckle, M., Geertz, M., Gualerzi, C., Mavathur, R., Muskhelshvili, G., Pon, C., Rimsky, S., Stella, S., Travers, A., (2007) High affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes. Nucleic Acids Res: 35(18): 6330-7. Geertz, M., Mavathur, R., Travers, A., Shimamoto, N., Muskhelishvili, G. RNA polymerase composition is homeostatically coupled with DNA topology in E. coli Submitted to EMBO reports. ABSTRACT Abstract Bacteria are omnipresent small organisms that posses impressive ability to adapt to their environment. Adaptation, a universal phenomenon seen at all different levels of organizational complexity of life, is accomplished mainly by adjustment of the gene expression pattern. Studies using microarray-mediated transcript profiling in E.coli demonstrated the existence of direct correlations between the global gene expression patterns and different phases [Tao, 1999; Weber, 2005; Wei, 2001]. Previous work from our and other laboratories indicated that in E.coli the cellular transcription is under the control of a global regulatory network whose main components are - DNA topoisomerases, chromatin proteins modulating the supercoil dynamics of DNA and, the RNA polymerase. The aim of this project was to understand the basic structural mechanism of interaction of two of the components (DNA supercoiling and chromatin proteins) and the underlying functional coordination of gene expression in E.coli. To understand the coordination of the components, the problem was approached at two levels of complexity, (1) global – for this purpose we developed a novel DNA microarray-based approach combining mutations in the genes of chromatin proteins with directional changes of DNA superhelicity and (2)local - at the level of individual gene promoter, using as a model tyrT promoter system, to understand the molecular mechanism of the interplay between supercoiling and transcriptional regulators. The study has led us to propose that the global transcription regulation is spatiotemporally coordinated by the differential distribution of two types of information, ‘analog’ and ‘digital’. The digital or discontinous property of an information is manifest in the pattern of expressed genes at all times depending on the distribution of the analog component - DNA supercoiling - distribution of supercoiling in different parts of the genome. The analog component is continuously converted in the pattern of expressed genes and vice versa, thereby modulates the physiology to a dynamic equilibrium, which can be readjusted in response to changes in both the digital (mutation) and analog (growth phase or stress induced changes of supercoiling) information. On the example of tyrT promoter we demonstrate how the sequence organization of the ABSTRACT promoter region determines the supercoiling sensitivity of the promoters. Our data indicate that the transcription from a strong promoter, like tyrT, is a consequence of two phenomena, the local effect of binding of chromatin protein FIS at the promoter region and the general effect of FIS on the average superhelical density of the DNA. ACKNOWLEDGEMENTS Acknowledgements I would like to express my appreciation, gratitude and thanks to the following people who have supported this work in many ways. I am indepted to Prof. Dr. Georgi Muskhelishvili not just for guiding my PhD work but for inspiring scientific temper in me, for being a mentor to whom I always looked up to during predicament and have very rarely been not satisfied by his suggestions and opinions to problems both scientific and general. Sincere thanks to Jacobs University Bremen (formerely International University Bremen) and Max Planck Institute for Terrestrial Microbiology at Marburg for giving me an opportunity to pursue my PhD work. I thank my doctoral examination committee, Prof. AndrewTravers, Prof. Albert Jeltsch and Prof. Sebastian Springer for support and motivation during my time at Jacobs University and for their thorough appraisal of this dissertation. I also take this opportunity to thank my family: my father, my mother, my sister Rohini, my borther-in-law Prakash, my nephew Varun, my niece Varsha, my brother Prasad, my sister-in-law Shvetha and my twin nieces Anagha and Anusha. The love showered by these people has always provided inspiration and motivation for me. A special thanks to my group members Marci, Michi, Sebastian, Nico and Claudia who made working in the lab an enjoyable experience. I will always cherish the time I spent with these intelligent people. And also thanks to Sissi for inducing and offering creative inputs on improving the appearance of my thesis and presentation beside being a good friend. Thanks also to all my friends in Marburg all of whom gave a very nice ACKNOWLEDGEMENTS first impression of Germany, so much so that I feel Marburg as my home town in Germany. The time at the university was pleasant because of my friends all of whom I wish to thank. And friends from IGC, Sanjay, Arun, Abhishek (more popularly known as ‘Badiya’) and Nandu for organizing and participating in the weekend parties. TABLE OF CONTENTS 1 Introduction ………………………………………………………………….. 1 1.1 Is there a common coordinating factor? ………………………………... 2 1.1.1 Is there a common coordinating mechanism? ………………………… 3 1.2 Global transcription coordination - current knowledge …………….. 4 1.2.1 Gene promoters ………………………………………………………. 5 1.2.2’Gene promoters respond distinctly to changes of supercoiling ………… 7 1.2.3 DNA Supercoiling and mechanisms regulating it ……………………. 9 1.2.4 Supercoiling and growth transitions …………………………………. 11 1.2.5 The nucleoid associated proteins – major players in global gene expression regulation …………………………………………………………….. 12 1.2.6 Composition of RNAP also changes with growth …………………… 14 1.3 Organisation of global transcription - coordinated view …………… 15 2 Materials and methods ……………………………………………………… 18 2.1 Materials ……………………………………………………………….. 18 2.1.1 Enzymes & chemicals ………………………………………………… 18 2.1.2 Antibiotics ……………………………………………………………. 19 2.1.3 Media …………………………………………………………………. 19 2.1.4 Buffers and stock solutions …………………………………………… 20 2.1.5 Bacterial strains ………………………………………………………. 21 2.1.6 Plasmids ……………………………………………………………… 22 2.1.7 Primers ……………………………………………………………….. 23 2.2 Methods ………………………………………………………………… 24 2.2.1 Agarose gel electrophoresis ………………………………………….. 24 2.2.2 Small and medium scale isolation of plasmid DNA using Qiagen kit … 24 2.2.3 Restriction Digestion ………………………………………………… 25 2.2.4 Polymerase chain reaction …………………………………………… 25 I TABLE OF CONTENTS 2.2.5 Preparation of competent E.coli cells using calcium chloride ……….. 25 2.2.6 Transformation of E.coli cells ………………………………………… 25 2.2.7 Purification of nucleic acids by Phenol:Chloroform extraction ……… 26 2.2.8 Concentrating and quantification of nucleic acids …………………… 26 2.2.9 Polyacrylamide sequencing gels ……………………………………… 26 2.2.10 Growth curves and norfloxacin treatment ……………………………. 27 2.2.11 Extraction and quality check of total RNA …………………………… 27 2.2.12 Microarray hybridization …………………………………………….. 28 2.2.13 Microarray analysis …………………………………………………… 29 2.2.14 Real-time PCR ……………………………………………………….. 30 2.2.15 High resolution agarose gel electrophoresis and determination of sigma (σ) ……………………………………………………………………….. 30 2.2.16 In vitro transcription …………………………………………………. 31 2.2.17 Primer extension ……………………………………………………… 32 2.2.18 Potassium permanganate footpriniting ……………………………….. 32 2.2.19 β-galactosidase assay ………………………………………………… 33 3 Results ……………………………………………………………………….. 34 3.1 Growth phase dependent global transcriptional regulation ………… 34 3.1.1 Growth of CSH50 and LZ strains ……………………………………. 34 3.1.2 A novel microarray based approach to derive growth phase-specific transcript profiles organized by supercoiling and chromatin proteins ………………………………………………………………………….. 36 3.1.3 Changes of supercoiling during growth ……………………………… 38 3.1.4 Genomic wheels of supercoiling sensitivity …………………………. 39 3.1.5 Variations of supercoiling-associated transcripts …………………….. 42 3.1.6
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