Genetic analysis of GcvB:yifK interaction unveils a translational enhancer in the target sequence

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Hfq: mediator of sRNA activity

In enteric bacteria, Hfq is a protein that participates in mRNA expression and stability and, as a chaperone, in sRNA-mediated regulation. Hfq was first identified as a host factor for RNA phage Qß replication in vitro (also known as host factor 1)(Franze de Fernandez et al., 1968, Franze de Fernandez et al., 1972, Miranda et al., 1997, Su et al., 1997). Phylogenetic analyses show that the hfq gene (or a related gene) is present in approximately half of all sequenced Gram-positive and Gram-negative genomes, with some bacteria harboring more then one hfq-like gene (Sun et al., 2002, Valentin-Hansen et al., 2004). Expression of these genes and functionality of their products remains to be demonstrated in most cases. Proteins of the Hfq family range from 70 to 110 amino acids and are generally organized in a homohexameric structure (Franze de Fernandez et al., 1972, Moller et al., 2002, Zhang et al., 2002). E. coli contains between 50,000 to 60,000 Hfq monomers (about 10,000 hexamers), the majority of which (80% to 90%) are found in the cytoplasmic fraction in association with ribosomes (Vasil’eva Iu & Garber, 2002). A significant amount is also located in the close proximity to the cytoplasmic membrane (Diestra et al., 2009, Vassilieva et al., 2002).
A main role of Hfq is to associate with trans-encoded sRNA and participate in the mechanism of post-transcriptional regulation. Thus, the protein is a direct regulator of genes under sRNA control, which include genes involved in stress responses (Muffler et al., 1997), virulence (Sittka et al., 2007, Ding et al., 2004) and quorum-sensing regulation (Meibom et al., 2009, Lenz et al., 2004). Furthermore Hfq plays a key role in the cellular response to phosphosugar toxicity and low iron levels (Fantappie et al., 2009, Gorke & Vogel, 2008, Vanderpool, 2007, Masse & Gottesman, 2002). Hfq also functions in cell envelope homeostasis through its role in the activity of MicA and RybB. These two sRNAs coordinately downregulate the expression of outer membrane proteins in stationary phase and under stress conditions (Bossi et al., 2008, Figueroa-Bossi et al., 2006a, Papenfort et al., 2006, Rasmussen et al., 2005).

Hfq structure and RNA binding patterns

Hfq is composed of 6 identical 11.2 kDa subunits. The stable, ringshaped structure of the hexamer was initially visualized by electron microscopy (Zhang et al., 2002). and subsequently resolved by x-ray crystallography, with or without synthetic RNA bound to it (Schumacher et al., 2002). The ring, about 70 Å in diameter, has a positively charged central pore on one of its two faces (Fig. 10). Studies with synthetic oligomers suggest that the two surfaces of the protein have different binding specificities: sequences rich in U residues bind preferentially to the proximal face, whereas Acontaining sequences bind to the distal face (Link et al., 2009, Mikulecky et al., 2004). It should be mentioned that even though the global architecture of the hexamer is conserved among species, some differences exist, in particular in the charge distribution. For instance, E. coli Hfq has a positive electrostatic surface for the trough that connects the proximal and distal faces, which is in contrast to the same area on the S. aureus Hfq, which shows a negative electrostatic surface (Fig. 10).

Mechanism of action of Hfq

The main role of Hfq in sRNA reguation is to stimulate the base-pair interaction between sRNAs and mRNAs (Fig. 11). Hfq was shown to increase the rate of sRNA-mRNA binding (Schumacher et al., 2002, Kawamoto et al., 2006) and to remodel RNA secondary structures (Geissmann & Touati, 2004, Moller et al., 2002). For example Hfq binding to the 5ʼ end of rpoS mRNA – to the sequence (AAN)4 – was shown to facilitate pairing between rpoS mRNA and DsrA sRNA (Soper & Woodson, 2008). The (AAN)4 sequence is also required in vivo for Hfq dependent regulation of rpoS translation by rpoS activating sRNAs in E. coli (Soper et al., 2010).

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Global sRNA-dependent regulatory networks: selected examples

Thanks to the ability to regulate multiple targets, trans-encoded sRNAs can mediate global regulatory responses. A paradigm for this type of response is provided by the RyhB sRNA, which controls iron homeostasis in E.coli and Salmonella. Iron is one of most important metal ions for bacteria, as it is used as a cofactor in many enzymes, such as those involved in the tricarboxylic acid (TCA) cycle, respiration and DNA synthesis. However, iron is also potentially toxic since it can react with oxygen to form free radicals that can damage cell components. Therefore, the concentration of free ferrous ion in all cells is tightly regulated. In E. coli and other bacteria, this regulation involves two factors: the Fur protein and RyhB sRNA. Fur is an iron-binding protein, that when bound to iron, represses transcription of genes involved in iron uptake (Hantke, 2001). Among the genes repressed by Fur is the ryhB gene (Masse & Gottesman, 2002). Like the other members of the Fur regulon, the ryhB gene is activated (derepressed) when iron becomes limiting. Upon accumulating, RybB downregulates the synthesis of a number of ironutilization and iron-storage proteins by pairing with sequences near the translation initiation sites of their mRNAs and inducing their degradation (Fig. 13) (Masse & Gottesman, 2002). In doing so, the sRNA limits iron consumption when the metal is scarce. As iron levels return to normal, ryhB repression by Fur is restored. Therefore, indirectly Fur upregulates all of the RybB-repressed genes, as many as 18 of which have been identified in E. coli (Masse et al., 2005).

Table of contents :

ABSTRACT
RESUMÉ
I. INTRODUCTION
I.1.Salmonella: general features
I.2. Gene Expression
I.2.1. Transcription
I.2.1.1. Alternative Sigma Factors
I.2.1.2.Transcriptional regulators
I.2.1.3. Transcription termination
I.3. Translation
I.3.1. S1 protein
I.3.2. Translation initiation factors
I.4. Global transcription regulatory networks: selected examples
I.4.1. The leucine response regulator Lrp
I.4.2. The glycine cleavage system
I.5. Regulation by RNA
I.5.1. Riboswitches
I.5.2. CRISPR systems
I.5.3. Small regulatory RNAs
I.5.3.1. Protein-targeting sRNAs
I.5.3.2. RNA-targeting sRNAs
I.5.3.2a. Cis-encoded sRNAs.
I.5.3.2b. Trans-encoded sRNAs
I.5.3.3. Hfq: mediator of sRNA activity
I.5.3.3a. Hfq structure and RNA binding patterns
I.5.3.3b. Mechanism of action of Hfq
I.5.3.4 Global sRNA-dependent regulatory networks:
selected examples
I.5.3.4a. RybB and iron homeostasis
I.5.3.4b. GcvB and amino acid management
II. RESULTS
II.1. Characterization of yifK, a Hfq-regulated locus in Salmonella
II.1.1. yifK is repressed by GcvB
II.1.2. Genetic analysis of GcvB:yifK interaction unveils a translational enhancer in the target sequence
II.1.3. Separate subdomains of GcvB R1 region pair with yifK and dppA
II.1.4. Role of ACA motifs in translation enhancement
II.2. Manuscript submitted to PloS Genetics
II.2.1 Supporting information of manuscript
II.3. An intriguing regulator of yifK expression
II.4. A specialized mutant ribosome improves initiation from a UUG start codon
II.5. Article II
III. DISCUSSION AND PERSPECTIVES
IV. MATERIALS AND METHODS
Table 2
Table 3
Table 4
V. REFERENCES

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