Please use this identifier to cite or link to this item: https://zone.biblio.laurentian.ca/handle/10219/2275
Title: Identifying Escherichia Coli factors that selectively bind the mRNA of secreted proteins
Authors: Hakeem, Walaa Abdulhadi H.
Keywords: mRNA;E. coli SRP;secreted proteins;amino acids;enzyme
Issue Date: 10-Nov-2014
Publisher: Laurentian University of Sudbury
Abstract: It is well accepted that the majority of secreted proteins are targeted to the secretory pathway through amino acid signal sequences located at the N–terminus of the pre-protein during the initial stages of translation on the ribosome. This is true in both eukaryotic and prokaryotic systems. These signal sequences display distinct structural features such as charged/hydrophilic residues at each termini and a continuous hydrophobic stretch of amino acids between them. The signal-recognition particle (SRP), which targets the ribosome to the membrane translocon for secretion of the protein, is hypothesized to recognize and these features and therefore differentiate these proteins from non-secretory proteins. Recent work in other laboratories has suggested a role for the mRNA itself, rather than only the amino acid sequence of the N–terminus of the pre-protein as playing a role in targeting the pre-protein-ribosome complex to the translocon within the membrane. To test this hypothesis, direct interaction between mRNAs encoding secreted proteins and the E. coli SRP equivalent (Ffh) was pursued using pull down assays. The mRNA’s used as bait corresponded to the N-terminal 40 amino acids of secreted and cytosolic proteins including periplasmic propyl isomerase chaperone SurA (as a model secretory protein with a cleavable signal peptide) and the cytoplasmic protein 3-isopropyl malate dehydrogenase (IsodH). Additionally, the mRNA of two other proteins, PhoA (secreted) and GMP (cytoplasmic), were used but in these mRNA the 5' UTR were also included in case these regions were involved in SRP recognition. Following extensive optimizations and modifications of these experiments, the Ffh protein (the Escherichia coli SRP homolog) could not be isolated from cytoplasmic extracts of E. coli with the pull down assays. One interesting finding however was that the mRNA of the IsodH protein was pulled down using its cognate mRNA transcript as bait. This implies a role for this enzyme in regulating its own levels in the cell by binding to and potentially modulating its translation. Other factors involved in DNA and RNA binding were also isolated and include RNase and ribosomal proteins, amongst others. It can therefore be concluded that under these experimental conditions, the mRNA hypothesis for targeting protein secretion could not be supported.
URI: https://zone.biblio.laurentian.ca/dspace/handle/10219/2275
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Master's Theses

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