Ultrastructure and complex polar architecture of the human pathogen Campylobacter jejuni

Campylobacter jejuni is one of the most successful food-borne human pathogens. Here we use electron cryotomography to explore the ultrastructure of C. jejuni cells in logarithmically growing cultures. This provides the first look at this pathogen in a near-native state at macromolecular resolution (∼5 nm). We find a surprisingly complex polar architecture that includes ribosome exclusion zones, polyphosphate storage granules, extensive collar-shaped chemoreceptor arrays, and elaborate flagellar motors

Structures of SRP54 and SRP19, the Two Proteins that Organize the Ribonucleic Core of the Signal Recognition Particle from Pyrococcus furiosus

In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 Å resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely α-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 Å resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19•SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP

A Computational Investigation on the Connection between Dynamics Properties of Ribosomal Proteins and Ribosome Assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins

Recognition of cognate transfer RNA by the 3OS ribosomal subunit

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 ang-stroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the uni-versally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the Þrst two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or Òwob-ble,Ó position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs. During protein synthesis, the ribosome cata-lyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as

Interactive stated choice surveys: a study of air travel behaviour

Stated preference (SP) experiments are becoming an increasingly popular survey methodology for investigating travel behaviour. Nevertheless, some evidence suggests that SP experiments do not mirror decisions in real markets. With an increasing number of real world decisions made using the internet, an opportunity exists to improve the realism of the SP counterparts of such choices by aligning the choice environment with such online portals. In this paper, we illustrate the benefits of such an approach in the context of air travel surveys. Our survey is modelled on the interface and functionality of an online travel agent (OTA). As with a real OTA, many ticket options are presented. Sort tools allow the options to be reordered, search tools allow options to be removed from consideration, and a further tool allows attributes to be hidden and shown. Extensive use of these tools is made by the 462 respondents. A traditional SP component was also completed by the respondents. Our exploratory analysis as well as random utility model estimation results confirm not only that respondents seem to engage more actively with the interactive survey, but also that the resulting data allows for better performance in model estimation compared to a more conventional SP experiment. These results have implications for the study of other complex travel choices where interactive surveys may similarly be preferable to standard approaches