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Bioinformatics, 31 (18), ISSN:1367-4803, ISSN:1460-2059

Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Proteins that bind single-stranded nucleic acids have crucial roles in cells, and structural analyses have contributed to a better understanding of their functions. In this issue of Structure, Dickey and colleagues describe several high resolution structures of a single OB-fold bound to different single-stranded DNA (ssDNA) sequences and reveal a spectacular co-adaptability of the protein/ssDNA interactions.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation., Author Summary Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillin. Among these bacteria some of them possess a beta-lactamase that is only produced at a high level when a beta-lactam antibiotic is present outside the cell. This mechanism of regulation, named beta-lactamase induction, has been described both in Gram-negative and Gram-positive bacteria. In Staphylococcus aureus and Bacillus licheniformis, two Gram-positive bacteria, their beta-lactamase is induced by homologous proteins including a membrane-bound penicillin receptor, BlaR1, and a cytoplasmic DNA-binding protein, BlaI, acting as repressor. The first step in the induction mechanism is the acylation of the extracytoplasmic domain of BlaR receptor by the beta-lactam antibiotic concomitant with the activation of the peptidase activity of its cytoplasmic domain. The activated receptor launched a cytoplasmic signal leading to the inactivation of BlaI repressor. The nature of this cytoplasmic signal is not well understood. In our study, we identified that a dipeptide issued from the bacterial cell wall is able to inactivate the BlaI repressor. It is the first time that a cell wall fragment is shown to reenter in the cytoplasm of Gram-positive bacteria to regulate gene expression.