Author(s): Apura, Patrícia de Faria Pais
Date: 2014
Persistent ID: http://hdl.handle.net/10362/12400
Origin: Repositório Institucional da UNL
Subject(s): Small RNAs; Gene expression; Synthetic biology; Transcriptional control; Post-transcriptional control
Dissertation presented to obtain the Master Degree in Molecular, Genetics and Biomedicine
Transcriptional and post-transcriptional control of gene expression dictate the levels of proteins in the cell. Therefore the modulation of gene expression can have important consequences for biotechnological and/or pharmaceutical purposes. Among the types of cellular RNAs, small RNAs (sRNAs) have been an emerging class of bacterial gene expression regulators, which mostly act by base-pairing with one or more mRNA target(s) affecting their translation and/or their stability. Here, we focus on the study of SraL sRNA, more specifically in the validation of putative targets for this sRNA obtained in a previous transcriptomic analysis. Until now SraL was only shown to regulate the mRNA levels of Trigger Factor, an important protein chaperone. The information here reported give strong evidence for SraL involvement in the cysteine biosynthetic pathway, which requires further investigation. Nevertheless, our results could not provide a validation of those putative targets previously obtained by transcriptomic analyses. Optimization of protein expression requires not only an increase of the stability of mRNA transcripts but also an optimal behavior of function-encoding DNA segments, which are often context-dependent. Building on the work of others, we have designed a set of combinatorial promoters and 5’UTRs and evaluated their effects/outcomes using Superfolder GFP as reporter. Our data shows a clear variability of protein levels within our set of constructs. The highest levels of protein were associated with the implementation of an insulation sequence flanking the promoter region and the introduction of 5’ stabilizing structures at the mRNA level. Further investigation concerning the alteration of the rate of the mRNA decay by depletion of the function of participating nucleases, might constitute an advantageous approach. The knowledge collected will be extremely important to design robust modules which substantially increase protein production. This field is rapidly growing and much remains to be discovered about these important regulatory processes.
