<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.

<p>Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein.</p><p>Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated <i>Escherichia coli</i> individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ.</p><p>Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA.</p><p>We have here compared how an SD⁺ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD⁺ is confirmed. A downstream SD⁺ gives decreased gene expression. If an SD⁺ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD⁺ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD⁺ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD⁺ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD⁺or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.</p>

Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein. Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated Escherichia coli individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ. Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD+ or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.