Genome-scale analyses of chromosomal accessibility, transcriptional initiation, and synthetic cross-feeding in Escherichia coli
By: by Nikos Basil Reppas
Published: 2007
Language: eng
Number of pages: 163
Description: Histone proteins in eukaryotes are known to restrict the accessibility of transcription factors to genomic DNA. Prokaryotes are thought to possess proteins of similar function, but the question of genomic accessibility has not been addressed. In Chapter 2, we combine chromatin immunoprecipitation (ChIP) in conjunction with high-density oligonucleotide microarray readout to define the in vivo genomic targets of the LexA transcriptional repressor. We show a near-universal relationship between the presence of a LexA sequence motif, LexA binding in vitro, and LexA binding in vivo, suggesting that a suitable recognition site for LexA is sufficient for binding in vivo. We also identify around 20 novel LexA targets that lack a canonical LexA sequence motif, precisely identifying the unusual 16 bp target sequence in one instance. In Chapter 3, using ChIP with fully tiled microarray readout, we perform a genome-wide analysis of the transition between transcriptional initiation and elongation in Escherichia coli by determining the association of core RNA polymerase (RNAP) and the promoter-recognition factor [sigma] 70 with respect to RNA transcripts. We identify over 1200 [sigma] 70 -associated promoters and demonstrate that [sigma] 70 is usually released very rapidly from elongating RNAP complexes in vivo. On average, RNAP density is higher at the promoter than in the coding sequence, although the ratio is highly variable among different transcribed regions. We show that the transition from transcriptional initiation to elongation is highly variable, often rate-limiting, and in some cases is essentially blocked such that RNAP is effectively poised to transcribe only under the appropriate environmental conditions.

In Chapter 4, we instantiate a model of biological cooperation using two strains of E. coli. One strain is a defective is tryptophan biosynthesis and the other in tyrosine biosynthesis; while each strain on its own is unable to grow without amino acid supplementation, the pair can be propagated together due to their mutualistic cross-feeding. We experimentally evolve this coculture, selecting for faster growth rates, and thus a higher degree of cooperation. We genetically reconstruct one of the evolved tryptophan auxotrophs that was subjected to whole genome sequencing, and fully recapitulate its improved growth phenotype.
Notes: Thesis (Ph.D., Committee on Higher Degrees in Biophysics)--Harvard University, 2007.

Includes bibliographical references.