Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.

Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5'-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

In-cell NMR spectroscopy in Escherichia coli.

A living cell is a complex system that contains many biological macromolecules and small molecules necessary for survival, in a relatively small volume. It is within this crowded and complex cellular environment that proteins function making in-cell studies of protein structure and binding interactions an exciting and important area of study. Nuclear magnetic resonance (NMR) spectroscopy is a particularly attractive method for in-cell studies of proteins since it provides atomic-level data noninvasively in solution. In addition, NMR has recently undergone significant advances in instrumentation to increase sensitivity and in methods development to reduce data acquisition times for multidimensional experiments. Thus, NMR spectroscopy lends itself to studying proteins within a living cell, and recently "in-cell NMR" studies have been reported from several laboratories. To date, this technique has been successfully applied in Escherichia coli (E. coli), Xenopus laevis (X. laevis) oocytes, and HeLa host cells. Demonstrated applications include protein assignment as well as de novo 3D protein structure determination. The most common use, however, is to probe binding interactions and structural modifications directly from proton nitrogen correlation spectra. E. coli is the most extensively used cell type thus far and this chapter is largely confined to reviewing recent literature and describing methods and detailed protocols for in-cell NMR studies in this bacterial cell.

The relationship between types of female athletic participation and female body type.

In the present study, the authors examined 53 female athletes drawn from 4 different sports and 55 female nonathletes. The athletes were divided into 2 groups: speed focused and technique focused. The nonathlete control group consisted of college women who had not participated in any varsity sports at the university level. Participants were measured on scales of body dissatisfaction, preoccupation with weight, and self-perceptions of body type and weight. Analyses revealed that (a) speed-focused athletes and technique-focused athletes did not differ significantly in their concerns about weight and body image, and (b) nonathletes expressed more dissatisfaction with their bodies than both of the athlete groups. Results are discussed with regard to associations between female sports participation and body image.