UC San Diego

In bacterial transcription, transcription initiation is arguably the most important regulatory point, because transcribing unnecessary genes into RNA could be a waste of energy, time and resources. There are multiple components which are involved in bacterial transcription initiation: RNA polymerase, [sigma]-factors, transcription factors, and transcription start sites. Each component has been intensively investigated, however in a limited scope and mostly with low-throughput methods. New technologies, such as hybridization on microarray and deep-sequencing, enabled researchers to study each component in a systems level, in a combination of two or more components, and in comparison between different species. In order to facilitate the analysis, integration, and comparison, software, MetaScope, was developed to accommodate multiple genome-scale datasets to visualize, analyze, integrate, and compare. TSS-seq, modified 5'-RACE with deep- sequencing, gave a genome-scale landscape of transcription start sites, and comparison of TSSs of conserved genes between closely-related species, E. coli and K. pneumoniae, showed significantly different usage of promoters, which implies different regulation of orthologous genes. To further investigate properties of promoters which were identified by TSS-seq, ChIP-chip experiments were performed for [sigma]-factors in E. coli to determine [sigma]-factor regulons. From the reconstructed [sigma]- factor network, extensive overlaps between regulons were observed. [sigma]⁷⁰ and [sigma]³⁸ share the largest set of genes in E. coli, and additional experiments revealed that those [sigma]-factors work in competition and utilize the negative regulation by [sigma]³⁸ . ChIP-exo, which applies exonuclease to present better resolution of DNA-binding, and RNA-seq implemented more detailed identification of Fur regulon in E. coli. Reconstruction of Fur regulon completed the previous knowledge of bacterial response to iron change, and also enabled its role over iron metabolism. In order to understand how bacteria respond to nitrogen limitation, the same methods were used under conditions that were predicted from model-based prediction, and resulted in reconstruction of regulons for major transcription factors, NtrC and Nac. Determination of those regulons expanded the current knowledge of nitrogen metabolism and how it is regulated in bacteria. Thus, systems approaches enabled a genome-scale assessment of regulatory components in multiple levels, and contributed to expansion of the current knowledge of bacterial transcription initiation