Diversity of LSM Family Proteins: Similarities and Differences.

Members of the Lsm protein family are found in all three domains of life: bacteria, archaea, and eukarya. They are involved in numerous processes associated with RNA processing and gene expression regulation. A common structural feature of all Lsm family proteins is the presence of the Sm fold consisting of a five-stranded ß-sheet and an α-helix at the N-terminus. Heteroheptameric eukaryotic Sm and Lsm proteins participate in the formation of spliceosomes and mRNA decapping. Homohexameric bacterial Lsm protein, Hfq, is involved in the regulation of transcription of different mRNAs by facilitating their interactions with small regulatory RNAs. Furthermore, recently obtained data indicate a new role of Hfq as a ribosome biogenesis factor, as it mediates formation of the productive structure of the 17S rRNA 3'- and 5'-sequences, facilitating their further processing by RNases. Lsm archaeal proteins (SmAPs) form homoheptamers and likely interact with single-stranded uridine-rich RNA elements, although the role of these proteins in archaea is still poorly understood. In this review, we discuss the structural features of the Lsm family proteins from different life domains and their structure-function relationships.

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization.

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Šresolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.

Overproduction and purification of the Escherichia coli transcription factors "toxic" to a bacterial cell.

Transcription factors play a crucial role in control of life of a bacterial cell, working as switchers to a different life style or pathogenicity. To reconstruct the network of regulatory events taking place in changing growth conditions, we need to know regulons of as many transcription factors as possible, and motifs recognized by them. Experimentally this can be attained via ChIP-seq in vivo, SELEX and DNAse I footprinting in vitro. All these approaches require large amounts of purified proteins. However, overproduction of transcription factors leading to their extensive binding to the regulatory elements on the DNA make them toxic to a bacterial cell thus significantly complicating production of a soluble protein. Here, on the example of three regulators from Escherichia coli, UxuR, ExuR, and LeuO, we show that stable production of toxic transcription factors in a soluble fraction can be significantly enhanced by holding the expression of a recombinant protein back at the early stages of bacterial growth. This can be achieved by cloning genes together with their regulatory regions containing repressor sites, with subsequent growth in a very rich media where activity of excessive regulators is not crucial, followed by induction with a very low concentration of an inducer. Schemes of further purification of these proteins were developed, and functional activity was confirmed.