Genome wide analyses of the Escherichia coli primary and secondary transcriptomes

Escherichia coli K12 serves as an important model for studying systems that are important to bacteria in their own right as well as those that are conserved in ‘higher' organisms, which are more difficult and costly to study. Like many model organisms, the genome of K12 has been sequenced, producing a catalogue of protein-coding and stable-RNA genes that enabled study using ‘omic’ approaches. This has led to a rapid expansion of our knowledge of patterns of gene expression and their dependency on growth conditions, cell physiology and individual genes. However, the underlying networks of gene regulation are less well understood, but are known to involve the control of steps in RNA processing and degradation as well as transcription and translation. With this in mind, this thesis describes the development of an approach based on RNA sequencing that produces nucleotide-resolution transcriptome maps that distinguish sites that correspond to RNA processing and steps in degradation from those of transcription initiation, while incorporating all classes of RNA. Comparison with results obtained previously validated the approach, which has been applied already to the study of other bacterial species. Within the E. coli map, many new features were identified, such as previously undetected small RNAs and processing at a site associated with the production of specialised ribosomes, which may ensure the translation of leaderless mRNAs, which were also mapped. The approach also showed the benefit of incorporating steps that can differentiate the 5’ status of transcripts in assigning sites of transcription initiation. RNA sequencing was also used to map sites of cleavage by RNase E, an essential endoribonuclease that is central to both the processing and degradation of RNA in bacteria and plant plastids. This aspect of the thesis has advanced from pilot studies to the point where the ‘code’ that determines one form of substrate recognition by RNase E is beginning to emerge. As a result of this success, equivalent data has been collected for other ribonucleases involved in RNA processing and degradation. Continuing analysis of the primary and secondary transcriptomes, consisting of native, unprocessed transcripts and of transcripts that have been modified from their native form via processing and/or degradation respectively, with the tools presented here promises to broaden and deepen our understanding of an important model organism.