Translation is a crucial factor in determining the rate of protein biosynthesis; for this reason, bacterial species typically evolve features to improve translation efficiency. Biosynthesis is a finely tuned cellular process aimed at providing the cell with an appropriate amount of proteins and RNAs to fulfill all of its metabolic functions. A key bacterial feature for faster recognition of the start codon on mRNA is the binding between the anti-Shine-Dalgarno (aSD) sequence on prokaryotic ribosomes at the 3’ end of the small subunit (SSU) 16S rRNA and Shine-Dalgarno (SD) sequence, a purine-rich sequence located upstream of the start codon in the mRNA. This binding helps to facilitate positioning of initiation codon at the ribosomal P site. This pairing, as well as factors such as the location of aSD binding relative to the start codon and the sequence of the aSD motif can heavily influence translation efficiency. The objective of this thesis is to understand the SD-aSD interactions and how changes in aSD sequences can affect SD sequences in addition to the underlying impact these changes have on the translational efficiency of prokaryotes. In chapter two, we hypothesized that differences in the prevalence of SD motifs between B. subtilis and E. coli arise as a result of changes in the free 3' end of 16S rRNA which may have led B. subtilis and E. coli to evolve differently. E. coli is expected to be more amenable to the acquisition of SD motifs that do not perfectly correspond with its free 3’ 16S rRNA end than B. subtilis. Further, we proposed that the evolutionary divergence of these upstream sequences may be exacerbated in B. subtilis by the absence of a functional S1 protein. Based on the differences between E. coli and B. subtilis, we were able to identify SD motifs that can only perfectly base pair in one of the two species and are expected to work well in one species, but not the other. Furthermore, we determine the frequency and proportion of these specific SD motifs that are expected to be preferentially present in one of the two species. Our motif detection is in keeping with the expectation that the predicted five categories of SD that are associated with B. subtilis and are expected to be less efficient in E. coli exhibit greater usage in the former than latter. Similarly, the predicted category of SD motifs associated with the E. coli 16S rRNA 3’ end is used more frequently in E. coli.Across prokaryote genomes, translation initiation efficiency varies due to codon usage differences whereas among genes, translation initiation varies because different genes vary in SD strength and location. In chapter 3 we hypothesized that there is differential translation initiation between 16 archaeal and 26 bacterial genomes. Translation initiation was found to be more efficient in Gram-positive than in Gram-negative bacteria and also more efficient in Euryarchaeota than in Crenarchaeota. We assessed the efficiency of translation initiation by measuring: i) the SD sequence’s strength and position and ii) the stability of the secondary structure flanking the start codon, which both affect accessibility of the start codon

<p>In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in <i>Escherichia coli</i>.</p><p>The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon.</p><p>The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR.</p><p>The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in <i>Escherichia coli</i>.</p>

In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in Escherichia coli. The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon. The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR. The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in Escherichia coli.