A Stress Response that Monitors and Regulates mRNA Structure Is Central to Cold Shock Adaptation.

Temperature influences the structural and functional properties of cellular components, necessitating stress responses to restore homeostasis following temperature shift. Whereas the circuitry controlling the heat shock response is well understood, that controlling the E. coli cold shock adaptation program is not. We found that during the growth arrest phase (acclimation) that follows shift to low temperature, protein synthesis increases, and open reading frame (ORF)-wide mRNA secondary structure decreases. To identify the regulatory system controlling this process, we screened for players required for increased translation. We identified a two-member mRNA surveillance system that enables recovery of translation during acclimation: RNase R assures appropriate mRNA degradation and the Csps dynamically adjust mRNA secondary structure to globally modulate protein expression level. An autoregulatory switch in which Csps tune their own expression to cellular demand enables dynamic control of global translation. The universality of Csps in bacteria suggests broad utilization of this control mechanism.

Translation efficiency is maintained at elevated temperature in Escherichia coli.

Cellular protein levels are dictated by the balance between gene transcription, mRNA translation, and protein degradation, among other factors. Translation requires the interplay of several RNA hybridization processes, which are expected to be temperature-sensitive. We used ribosome profiling to monitor translation in Escherichia coli at 30 °C and to investigate how this changes after 10-20 min of heat shock at 42 °C. Translation efficiencies are robustly maintained after thermal heat shock and after mimicking the heat-shock response transcriptional program at 30 °C by overexpressing the heat shock σ factor encoded by the rpoH gene. We compared translation efficiency, the ratio of ribosome footprint reads to mRNA reads for each gene, to parameters derived from gene sequences. Genes with stable mRNA structures, non-optimal codon use, and those whose gene product is cotranslationally translocated into the inner membrane are generally less highly translated than other genes. Comparison with other published datasets suggests a role for translational elongation in coupling mRNA structures to translation initiation. Genome-wide calculations of the temperature dependence of mRNA structure predict that relatively few mRNAs show a melting transition between 30 and 42 °C, consistent with the observed lack of changes in translation efficiency. We developed a linear model with six parameters that can predict 38% of the variation in translation efficiency between genes, which may be useful in interpreting transcriptome data.

Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σs as well as the specificity of their interaction with anti-σs. DNA synthesis was used to build 86 ECF σs (two from every subgroup), their promoters, and 62 anti-σs identified from the genomes of diverse bacteria. A subset of 20 σs and promoters were found to be highly orthogonal to each other. This set can be increased by combining the -35 and -10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal σs, anti-σs, and promoters were used to build synthetic genetic switches in Escherichia coli. This represents a genome-scale resource of the properties of ECF σs and a resource for synthetic biology, where this set of well-characterized regulatory parts will enable the construction of sophisticated gene expression programs.

Operon mRNAs are organized into ORF-centric structures that predict translation efficiency.

Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF.

CTREC: C-arm Tracking and Reconstruction using Elliptic Curves.

C-arm fluoroscopy is ubiquitous in contemporary surgery, but it lacks the ability to accurately reconstruct three-dimensional information, attributable to the difficulty in obtaining the pose of X-ray images in 3D space. We propose a unified mathematical framework to address the issues of intra-operative pose estimation, correspondence and reconstruction, using simple elliptic curves. In contrast to other fiducial-based tracking methods, our method uses a single ellipse to constrain 5 out of 6 degrees of freedom of C-arm pose, along with randomly distributed unknown points in the imaging volume (either naturally present or induced by randomly placed beads or other markers in the image space) from two images/views to completely recover the C-arm pose. Preliminary phantom experiments indicate an average C-arm tracking accuracy of 0.51° and 0.12° STD. The method appears to be sufficiently accurate and appealing for many clinical applications, since it uses a simple elliptic fiducial coupled with patient information and has very minimal interference with the workspace.