| dc.description.abstract | The information flow through the regulatory networks in biological systems has been a rapidly growing field of research. Translation, being a very important regulatory check point, presents itself as a legitimate process for investigation. Only few regulatory factors and pathways are currently delineated that regulate translation through intermediary components in a remote manner, with global implications. In this context, this thesis studies the proteomics and transcriptomics data of Escherichia coli (E. coli) mutants, defective in translation, with the aim to unravel such regulatory factors or pathways and thus probe their regulatory aspects.
Two main mics techniques are the backbone of this study; proteomics and transcriptomics. These provide a holistic view of cell states which allow us to investigate the regulation happening at the translation as well as at the transcription level. Two different proteomics techniques are used to resolve the proteomes; two-dimensional gel electrophoresis (2DE) and LC-coupled mass spectrometry. These have been introduced in the first chapter.
Transcriptomics and proteomics being an evolving field, most of the techniques need optimization before applied for actual experiments and data acquisition. As part of our experimental strategy, we performed both transcriptomics and proteomics experiments in parallel. During application of 2DE based proteomics, we observed significant deficiencies in the 2DE technique itself, which we addressed as our first priority. We ran numerous optimization protocols to arrive at an optimized protocol to remove acidic region streaking (ARS) in 2DE, which is a well-known artifact. We describe the development of the modified protocol and discuss the detailed comparative analyses with recently published 2DE gels confirming the efficacy of the method in Chapter 2.
The optimized 2DE technique developed by us was exploited in combination with MALDI mass spectrometry for the comparative proteomic analysis between the wild type
E. coli and a mutated (ΔmetZWV::kan) strain. The proteomics results and its functional validation revealed a direct link between the flux of 10-formyltetrahydrofolate and the regulation of purine metabolism. The experimental observations were computationally modelled using flux balance analysis to understand the mechanistic detail involved in the remote regulation driven by purine metabolism and other peripheral pathways. The experimental details and the computational modelling are covered in chapter three.
To gather wider perspective on the regulatory links in the E. coli organism, related to translation, we extended the omics studies using microarray technique on newer mutant strains. Our experiments aimed at obtaining differential transcript levels in the whole cell and the polysomal fraction of the E. coli cells. Three different E. coli mutants were used in this study; infC135, PthTs and folD122, which were defective in translation initiation, recycling and one carbon metabolism, respectively. The analysis revealed important routes of metabolic regulation. Few of them are worth mentioning; for example, purine and 10-fTHF metabolism that controls macromolecular synthesis, energy generation and inter-conversion of metabolites through pyruvate and also flagellar biosynthesis which is remote to translation. Transcriptomics data available from GEO database was analyzed as a background and based on the analysis we propose which of the differentially expressed genes are of generic in nature or unique to our mutants. These interesting observations about regulatory pathways are discussed in chapter four.
To validate our transcriptomics results at the proteomics level and with a higher sensitivity than 2DE proteomics, we studied the whole cell proteomics data from two E. coli mutants, infC135 and PthTs, using high resolution FT-ICR mass spectrometry. Although a small number of differentially expressed proteins compared to microarray data, we could correlate the results with our transcriptomics data, especially, the proteins in the catabolic pathways. We elaborate the aforesaid study in chapter five.
At the end we summarize the above omics studies to notice the following aspects emerging out. Translation, being a fundamental and essential process for the cell, disturbing it from any angle should affect many other processes which might seem remotely or not at all related to protein synthesis. This is evident from the whole study; we have been able to see some regulations which are very close to translation, but most are not directly related to translation. Apart from this we were able to point out routes of regulation which might control the amount of macromolecules synthesized, utilization of energy and metabolites and flagellar biogenesis. Another aspect is that we were able point out the gap in information between our regulation of pathways close to and remote to protein biosynthesis. Lastly, few master regulators were pointed out which might have potential functions in addition to what is known till date. A concluding discussion about these aspects has been discussed in the sixth chapter. | en_US |
