UNIVERSITY OF CALIFORNIA, SAN DIEGO Systems Evaluation of Regulatory Components in Bacterial Transcription Initiation A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Donghyuk Kim Committee in charge: Professor Bernhard Ø. Palsson, Chair Professor Eric E. Allen Professor Victor Nizet Professor Shankar Subramaniam Professor Kun Zhang 2014 Copyright Donghyuk Kim. 2014 All rights reserved The dissertation of Donghyuk Kim is approved, and it is acceptable in quality for publication on microfilm: _____________________________________________ _____________________________________________ _____________________________________________ _____________________________________________ _____________________________________________ Chair University of California, San Diego 2014 iii Dedication To Sejin Koo For your enduring love, and support. You truly are my best friend and a life-long companion of faith. To Isabella For your heart-warming smiles. May you live in goodness and faith. iv Epigraph 我非生以知之者, 好古, 敏以求之者也 孔子 It is the glory of God to conceal a matter, but the glory of kings is to search out a matter. Proverbs 25:2 v Table of Contents Dedication ................................................................................................................................. iv Epigraph ..................................................................................................................................... v Table of Contents ...................................................................................................................... vi List of Figures and Tables ......................................................................................................... ix Acknowledgements .................................................................................................................. xii Vita ........................................................................................................................................... xv Abstract of the Dissertation .................................................................................................... xvii Chapter 1: Genome-wide assessment of transcriptional regulatory components ....................... 1 Phenotype-genotype relationship ........................................................................................... 1 Regulatory components in transcription initiation ................................................................. 2 Experimental approaches to investigate regulatory components ........................................... 4 Chapter 2: MetaScope: a genome browser with embedded functions for analysis and integration of multiple omics datasets ........................................................................................ 7 Implementation ....................................................................................................................... 8 Overview ................................................................................................................................ 8 Streamlined workflow ............................................................................................................ 9 Performance ......................................................................................................................... 11 Chapter 3: Comparison of bacterial regulatory elements by transcription start site profiling.. 12 Experimental identification of TSSs in E. coli and K. pneumoniae ..................................... 14 Identification of small RNAs in K. pneumoniae .................................................................. 15 Similar usage of regulatory features ..................................................................................... 19 Different organization of the upstream regulatory region .................................................... 25 Comparison of regulatory non-coding small RNAs ............................................................. 29 Disucssion ............................................................................................................................ 34 Methods ................................................................................................................................ 39 Acknowledgements .............................................................................................................. 45 Chapter 4: Genome-scale reconstruction of the sigma factor network in Escherichia coli: topology and functional states .................................................................................................. 47 vi Determination of the genome-wide map of holoenzyme binding ........................................ 47 Determination of the genome-wide promoter map .............................................................. 50 Reconstruction of sigma factor regulons and their overlaps ................................................ 51 Sigma factor competition in overlapped promoters ............................................................. 53 Comparative analysis of the sigma factor network in close related species ......................... 56 Conclusions .......................................................................................................................... 60 Methods ................................................................................................................................ 60 Acknowledgements .............................................................................................................. 67 Chapter 5: Deciphering the Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli ............................................................................ 68 Genome-wide identification of Fur binding sites ................................................................. 70 Genome-wide reconstruction of Fur regulon ....................................................................... 72 Regulatory modes of Fur in response to iron availability .................................................... 74 Fur-regulated feedback loop motifs for iron metabolism ..................................................... 76 Elucidation of complex roles of Fur regulatory network beyond iron metabolism .............. 80 Discussion ............................................................................................................................ 84 Methods ................................................................................................................................ 86 Acknowledgements .............................................................................................................. 89 Chapter 6: Elucidating transcriptional regulation of nitrogen metabolism with systems approaches ................................................................................................................................ 90 Model-driven prediction of activation conditions for TFs ................................................... 91 Experimental confirmation of predicted conditions ............................................................. 93 Advantages of ChIP-exo over previous methods ................................................................. 95 Confirmation of binding sites with comparison to known sites ......................................... 100 Reconstruction of NtrC and Nac regulons ......................................................................... 101 Association of TF with σ-factor ......................................................................................... 103 Contrasting roles of NtrC and Nac regulons ...................................................................... 105 Primary response by NtrC regulon ..................................................................................... 107 Carbon flux rebalancing by Nac regulon ........................................................................... 110 Less activation of Nac on cytidine ..................................................................................... 113 Acknowledgements ............................................................................................................ 115 vii Chapter 7: Towards a detailed understanding of bacterial transcription regulation with systems approaches .............................................................................................................................. 116 Increasing needs for bioinformatic tools. ........................................................................... 116 Comparative systems biology on regulatory elements by genome-wide TSS profiling .... 117 Genome-scale reconstruction of σ-factor network in E. coli .............................................. 118 Systems determination of Fur transcriptional regulatory network ..................................... 119 Elucidating transcriptional regulation of nitrogen metabolism .......................................... 120 Conclusion .......................................................................................................................... 121 References .............................................................................................................................. 122 viii List of Figures and Tables Figure 1. Genotype-Phenotype relationship ..............................................................................