Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions.

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

The dynamic cycle of bacterial translation initiation factor IF3.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

Handheld computers for self-administered sensitive data collection: a comparative study in Peru.

BACKGROUND: Low-cost handheld computers (PDA) potentially represent an efficient tool for collecting sensitive data in surveys. The goal of this study is to evaluate the quality of sexual behavior data collected with handheld computers in comparison with paper-based questionnaires. METHODS: A PDA-based program for data collection was developed using Open-Source tools. In two cross-sectional studies, we compared data concerning sexual behavior collected with paper forms to data collected with PDA-based forms in Ancon (Lima). RESULTS: The first study enrolled 200 participants (18-29 years). General agreement between data collected with paper format and handheld computers was 86%. Categorical variables agreement was between 70.5% and 98.5% (Kappa: 0.43-0.86) while numeric variables agreement was between 57.1% and 79.8% (Spearman: 0.76-0.95). Agreement and correlation were higher in those who had completed at least high school than those with less education. The second study enrolled 198 participants. Rates of responses to sensitive questions were similar between both kinds of questionnaires. However, the number of inconsistencies (p = 0.0001) and missing values (p = 0.001) were significantly higher in paper questionnaires. CONCLUSION: This study showed the value of the use of handheld computers for collecting sensitive data, since a high level of agreement between paper and PDA responses was reached. In addition, a lower number of inconsistencies and missing values were found with the PDA-based system. This study has demonstrated that it is feasible to develop a low-cost application for handheld computers, and that PDAs are feasible alternatives for collecting field data in a developing country.