Despite their name, a number of the cold shock proteins are expressed during normal growth, and not just during cold shock, in several species. The function of these constitutively expressed CspA paralogues is unclear. In Salmonella Typhimurium (a major worldwide cause of gastrointestinal disease) they have been linked to various stress responses and the establishment of virulence. Study of the cold shock proteins as gene regulators is therefore of great interest, and they also have potential as targets for antimicrobial development. CspE in Salmonella Typhimurium is constitutively expressed during normal growth. In order to determine its function, attempts were made to identify the interactions it forms with other cellular proteins. Initially, a proteomic investigation attempted to identify proteins which complex with CspE by in vivo cross-linking and affinity purification followed by mass spectrometry. Although no defined complex was consistently identified, the results suggested a handful of proteins which might interact with CspE in a weak or transient manner. These proteins included many from the nucleoid and ribosomal entry site, hinting at CspE’s cellular localisation. In order to investigate these transient interactions, a bacterial two-hybrid system was employed. Interactions between CspE and HupA, a nucleoid protein identified in the proteomic analysis, were probed, as were interactions between CspE and CsdA, an RNA helicase thought to function co-operatively with CspE. The twohybrid system also allowed investigation of CspE dimerisation, which has been reported in vitro but not investigated in vivo until this study. CspE appears not to interact significantly with either HupA, CsdA, or itself at 37oC. Finally in a further attempt to identify interactions of CspE, a genomic library was created to test CspE interactions by two-hybrid assay with random peptides derived from the whole Salmonella genome. The library was successfully created and screened for evidence of interaction, and revealed an association between CspE and a transcriptional repressor, DeoT. DeoT is a repressor of several genes for catabolic processes, suggesting a role for CspE in the regulation of central metabolism. The findings of this work present a number of novel discoveries and several interesting opportunities for further studies.

Numerous essential cellular processes, such as gene regulation and tRNA processing, are carried out by structured RNAs. While in vitro most RNAs become kinetically trapped in non-functional misfolded states that render them inactive on a biologically-relevant time scale, RNAs folding in vivo do not share this same outcome. RNAs do indeed misfold in the cell; however, chaperone proteins promote escape from these non-native states and foster folding to functional conformations. DEAD-box proteins are ATP-dependent RNA chaperone proteins that function by disrupting structure, which can facilitate structural conversions. Here, studies with both local and global focuses are used to uncover mechanistic features of DEAD-box proteins CYT-19 and Mss116p. Both of these proteins are general RNA chaperones as they each have the ability to facilitate proper folding of multiple structured RNAs. The first study probes how DEAD-box proteins interact with a simple duplex substrate. Separating the strands of a duplex is an ATP-dependent process and is central to structural disruption by DEAD-box proteins. Here, how ATP is utilized during duplex separation is monitored by comparing ATP hydrolysis rates with strand separation rates. Results indicate that one ATP molecule is sufficient for complete separation of a 6-11 base pair RNA duplex. Under some conditions, ATP binding in the absence of hydrolysis is sufficient for duplex separation. Next, focus is shifted to a more global perspective as the function of Mss116p is probed in the folding of a cognate group II intron substrate, aI5[gamma], under near-physiological conditions. Three catalytically-active constructs of aI5[gamma] are used and catalysis serves as a proxy for folding. Folding of all constructs is promoted by the presence of Mss116p and ATP. In vitro and in vivo results indicate that a local unfolding event is promoted by Mss116p, stimulating formation of the native state. Lastly, orthogonal methods that probe physical features of RNA are used to provide insight into the structural intermediates with which Mss116p acts. / text

<p>Abstract</p><p> Hfq is a pleiotropic posttranscriptional regulator and RNA chaperone that facilitates annealing of trans-encoded sRNA/mRNA pairs. It regulates many different cellular pathways including environmental stress responses, quorum sensing, virulence and maintenance of membrane integrity. Hfq is a member of the Sm/LSm family and forms a homohexamer that has two faces, termed proximal and distal. Hfq preferentially binds A/U rich regions that are near stem loop structures. Crystal structures have shown that poly-A sequences tend to bind the distal face while poly-U sequences bind the proximal face. Currently crystal structures reveal the binding mechanisms for short RNA sequences however; physiologically relevant RNA sequences are typically longer and more structured. To study how these more complex RNA sequences interact with Hfq, a tryptophan fluorescence quenching (TFQ) assay has been developed. Here it is presented that TFQ can correctly identify the binding face for two control sequences, A15 and U6, using the E. coli, S. aureus and L. monocytogenes Hfq homologues. Using fluorescence anisotropy and crystallography it is observed that Trp mutants necessary for TFQ may affect binding to some degree but do not affect the overall structure or RNA binding function of Hfq. TFQ is then used to examine the distal face binding motifs for both Gram-negative (E. coli) and Gram-positive (S. aureus/L. monocytogenes) Hfq, (A-R-N)n and (R-L)n respectively. Using sequences that either fulfilled just (A-R-N)n or both (A-R-N)n and (A-A-N)n motifs it is shown that the distal face motif for Gram-negative Hfq is the more specific (A-A-N)n motif. Using sequences that either fulfilled just (R-L)n or both (R-L)n and (A-L)n motifs it is shown that the Gram-positive distal face motif can be redefined to the (A-L)n motif. Finally TFQ is used to explore autoregulation of E. coli hfq. Two identified binding sites located in the 5'UTR of hfq mRNA, site A and site B, were used for TFQ, along with a longer RNA sequence that contains both sites and their native linker, 5' UTR. TFQ illustrates that the individual sites and the 5' UTR are capable of binding both faces. Each site appears to prefer binding to one face over the other, suggesting a model for hfq 5' UTR mRNA binding to Hfq where either one or two hfq mRNA bind a single Hfq hexamer. In conclusion, TFQ is a straightforward method for analyzing how RNA sequences interact with Hfq that can be utilized to study how longer, physiologically relevant RNA sequences bind Hfq.</p> / Dissertation

<p>Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated.</p><p>In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype.</p><p>The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome.</p><p>In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.</p>

Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated. In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype. The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome. In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.

Le VIH-1 bourgeonne sous forme immature et doit subir l’étape de maturation afin d’acquérir son caractère infectieux. La maturation protéolytique du précurseur Pr55Gag induit le réarrangement morphologique de la particule alors que le dimère d’ARNg acquiert une compaction optimale. Ces réarrangements conformationnels restent encore inconnus et sont facilités par l’activité chaperonne de la protéine NCp7. Notre but a été de déterminer les différentes étapes menant à l’obtention d’un dimère d’ARNg mature. Nous avons donc étudié la structure des 550 premiers nucléotides du génome par cartographie chimique, à la fois 1. in vitro en présence des protéines Pr55Gag, GagΔp6, intermédiaires contenant le domaine NC et NCp7 et 2. in viro par l’approche hSHAPE-Seq que nous avons développé. Les particules matures et bloquées aux différentes étapes de maturation de Pr55Gag ont été analysées ainsi que des particules matures et totalement immatures traitées avec l’éjecteur de zinc AT-2. Ce traitement permet d’identifier les sites de protection de Pr55Gag et NCp7 ainsi que leur activité déstabilisatrice. / The HIV-1 particle buds from the infected cell as an immature particle and has to undergo a maturation process to become infectious. Proteolytic processing of Pr55Gag triggers morphological rearrangements of the particle whereas the gRNA dimer becomes more stable. Genomic rearrangements remain poorly understood and are facilitated by the RNA chaperone activity of the NCp7 protein. Our goal was to determining the different steps leading to the formation of the mature dimeric gRNA. To this end, the structure of the first 550 nucleotides of the HIV-1 genome was assessed by chemical probing 1. in vitro with Pr55Gag, GagΔp6, NC-containing intermediates and NCp7 proteins and 2. in viro with the hSHAPE-Seq approach we developed. Wild type and mutant viruses mimicking the sequential processing of Pr55Gag were analysed, as well as immature PR- and mature particles treated with the AT-2 zinc ejector, in order to identify the Pr55Gag and NCp7 binding sites and their gRNA destabilising activity.