Discrimination of 5′-terminal start codons by translation initiation factor 3 is mediated by ribosomal protein S1

AbstractThe interrelation between ribosomal protein S1 and IF3 in recognition/discrimination of 5′-terminal start codons by 30S ribosomes has been studied using in vitro toeprinting. The study has been performed with two naturally occurring leaderless mRNAs, λ cI and phage r1t rro mRNA, as well as with an artificial leaderless mRNA derived from the E. coli ompA gene. We show that in the absence of S1, IF3 does not discriminate against the authentic 5′-terminal start codon of both cI and rro mRNA. Since IF3 was able to exert its proofreading function for initiator tRNAMetf on 30S ribosomes lacking S1, this observation cannot be attributed to a lack of binding to or action of IF3 on 30S(−S1) ribosomes. In contrast to leaderless mRNAs, ternary complex formation occurs in the presence of IF3 with 30S ribosomes when the start codon is preceded by a short 20-nucleotide 5′-untranslated region containing a canonical Shine and Dalgarno sequence. This suggests that 5′-terminal start codons are recognised by IF3 as non-standard because of the lack of 16S rRNA-mRNA contacts

Stabilization of Hfq-mediated translational repression by the co-repressor Crc in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery

Harnessing Metabolic Regulation to Increase Hfq-Dependent Antibiotic Susceptibility in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for ~ 10% of hospital-acquired infections worldwide. It is notorious for its high level resistance toward many antibiotics, and the number of multi-drug resistant clinical isolates is steadily increasing. A better understanding of the molecular mechanisms underlying drug resistance is crucial for the development of novel antimicrobials and alternative strategies such as enhanced sensitization of bacteria to antibiotics in use. In P. aeruginosa several uptake channels for amino-acids and carbon sources can serve simultaneously as entry ports for antibiotics. The respective genes are often controlled by carbon catabolite repression (CCR). We have recently shown that Hfq in concert with Crc acts as a translational repressor during CCR. This function is counteracted by the regulatory RNA CrcZ, which functions as a decoy to abrogate Hfq-mediated translational repression of catabolic genes. Here, we report an increased susceptibility of P. aeruginosa hfq deletion strains to different classes of antibiotics. Transcriptome analyses indicated that Hfq impacts on different mechanisms known to be involved in antibiotic susceptibility, viz import and efflux, energy metabolism, cell wall and LPS composition as well as on the c-di-GMP levels. Furthermore, we show that sequestration of Hfq by CrcZ, which was over-produced or induced by non-preferred carbon-sources, enhances the sensitivity toward antibiotics. Thus, controlled synthesis of CrcZ could provide a means to (re)sensitize P. aeruginosa to different classes of antibiotics

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind

YfiK from Escherichia coli Promotes Export of O-Acetylserine and Cysteine

yfiK was discovered as a gene augmenting cysteine production when it was overexpressed in an industrial Escherichia coli production strain. The gene product is an integral membrane protein with about six predicted transmembrane helices; it belongs to the RhtB family of export proteins. YfiK overproduction from a plasmid leads to drastic and parallel secretion of O-acetylserine and cysteine into the medium but only when the organism possesses a serine transacetylase that is feedback insensitive to cysteine. Externally provided O-acetylserine obviated this requirement for cysteine secretion both in the yfiK-carrying transformant and in the wild type. A ΔyfiK mutant did not show any phenotype, and it exported O-acetylserine and cysteine when transformed with a plasmid carrying ydeD, a previously characterized, alternate O-acetylserine/cysteine exporter. Since a ydeD-yfiK double mutant showed the same pattern, it appears that YfiK and YdeD act independently. The necessity for the cell to regulate the size of the internal pool of O-acetylserine via synthesis of exporter proteins could be connected to the fact that this compound (when supplied externally) inhibits growth. Overexpression of either ydeD or yfiK leads to alleviation of this inhibition paralled by increased resistance to azaserine, which is an analog of O-acetylserine

MEASUREMENT-BASED MODELING OF END-TO-END DELAYS IN WLANS WITH NS-2 Abstract

We enhance the standard ns-2 model for IEEE 802.11 WLANs such that it reflects the delays that are measured in a real ad-hoc network composed of laptops. From timestamps for UDP transmissions, which are recorded at several points on the way from application layer to application layer in a lowly-loaded system, we derive various empirical distributions, which we incorporate into the ns-2 model in order to better capture the real system. For validation, we compare simulated and measured end-to-end delays for UDP transmissions under different load conditions. Our experiments demonstrate the difficulties encountered when tuning a generic and widely used simulation model to a real scenario and suggest that cross-correlation among subsequent delays need to be considered for a satisfactory match of simulation and measurements.

Scapular dyskinesis after Latarjet procedure

BACKGROUND: Because of detachment of the pectoralis minor and variation of the vector of the conjoint tendons, we hypothesized that the Latarjet procedure may alter scapular position and motion. The purpose of this study was to evaluate scapular position and motion in patients who underwent a Latarjet or a modified iliac crest bone graft transfer (ICBGT) procedure (J-bone graft). METHODS: Forty-six consecutive patients treated for recurrent anterior shoulder dislocation between 2010 and 2012 were retrospectively enrolled. Twenty-three were treated with a Latarjet and 23 with an ICBGT procedure. Twenty Latarjet and 20 ICBGT patients were available at a mean follow-up of 20 months (min, 12; max, 60). We recorded the Western Ontario Instability Index, the Rowe Score, and the Subjective Shoulder Value. Scapulothoracic position was studied according to the dyskinesis yes/no method. Intraobserver and interobserver reliability of the dyskinesis assessment was assessed. RESULTS: Intraobserver and interobserver reliability of scapula dyskinesis assessment was high (Latarjet: intratester, κ = 0.84; intertester, κ = 0.75; ICBGT: intratester, κ = 0.78; intertester, κ = 0.71). Scapular dyskinesis was observed after 5 of 20 Latarjet and after 0 of 20 ICBGT procedures (P = .047). Patients with dyskinesis had lower scores (Western Ontario Instability Index, P = .043; Rowe, P = .047; Subjective Shoulder Value, P = .046), but no statistically significant difference was found between the Latarjet and ICBGT groups. Two of the 5 scapular dyskinesis patients reached the SICK (Scapular malposition, Inferior medial scapular winging, Coracoid tenderness, and scapular dysKinesis) scapula syndrome definition. CONCLUSIONS: Scapular dyskinesis was found in 5 of 20 patients who underwent a Latarjet procedure. Dyskinesis may be related to the detachment of the pectoralis minor, and variation of the vector and the working length of the coracobrachialis and the short head of the biceps