Part I: Translation of leaderless mRNA in E. coli Spedalieri G., Kashrer C., RajBhandary U.L. Leaderless mRNAs are present in all three domains of life. They do not have a 5' untranslated region (5'UTR), they start directly with the initiation codon, AUG in most cases. In Escherichia coli these mRNAs are very rare and show inefficient translation. In contrast, leaderless mRNAs are quite prevalent in archaea, where they are translated as efficiently as the ''leadered'' mRNAs containing the typical Shine-Dalgarno sequence. In leaderless translation initiation 70S ribosomes are thought to be used without a typical pre-initiation complex involving a 30S ribosomal subunit. In this study, the goal is to answer two fundamental questions: (i) Is AUG strictly required as initiation codon? (ii) Is the formylmethionyl-tRNAfMet (fMet-tRNAfMet) required as initiator tRNA? Is the amino acid ''methionine'' important? Using a chloramphenicol acetyltransferase reporter system for studying in vivo translation of leaderless mRNAs in E. coli, we have found that the initiation codon AUG is not absolutely required and the amino acid ''methionine'' plays a fundamental role. Part II: Structural and functional characterization of cspD mRNA in Escherichia coli Gaetana Spedalieri, Anna Brandi and Anna Maria Giuliodori Expression of cspD, a gene belonging to the csp family of Escherichia coli, is exclusively induced at 37Â°C during stationary-phase or upon glucose starvation and does not increase during cold-shock. To shed light on the mechanisms responsible for the regulation of cspD expression, I have elucidated by enzymatic and chemical probing the secondary structure of cspD mRNA as a function of temperature. The structure, which was also validated by mutagenesis, demonstrates that cspD mRNA does not undergo any temperature-dependent structural rearrangement. Functional analyses suggest that the presence of a helix occluding the initiation codon might be partly responsible for the scarce translation of cspD mRNA and that RNase III is likely involved in the regulation of cspD gene expression. Overall, these data suggest that induction of cspD, like that of many other csp gene of E. coli, is controlled also at a posttranscriptional level.
|Titolo:||Translation of Leaderless mRNAs and Structure of cspD mRNA in E. coli|
|Data di pubblicazione:||16-apr-2010|
|Appare nelle tipologie:||Tesi di dottorato (Pregresso)|