RNA single strands bind to a conserved surface of the major cold shock protein in crystals and solution

Abstract

Bacterial cold shock proteins (CSPs) regulate the cellular response to temperature downshift. Their general principle of function involves RNA chaperoning and transcriptional antitermination. Here we present two crystal structures of cold shock protein B from Bacillus subtilis (Bs-CspB) in complex with either a hexanucleotide (59-UUUUUU-39) or heptanucleotide (59-GUCUUUA-39) single-stranded RNA (ssRNA). Hydrogen bonds and stacking interactions between RNA bases and aromatic sidechains characterize individual binding subsites. Additional binding subsites which are not occupied by the ligand in the crystal structure were revealed by NMR spectroscopy in solution on Bs-CspBÁRNA complexes. Binding studies demonstrate that Bs-CspB associates with ssDNA as well as ssRNA with moderate sequence specificity. Varying affinities of oligonucleotides are reflected mainly in changes of the dissociation rates. The generally lower binding affinity of ssRNA compared to its ssDNA analog is attributed solely to the substitution of thymine by uracil bases in RNA.

Mechanisms of Lin28-Mediated miRNA and mRNA Regulation—A Structural and Functional Perspective

“…Binding of ssDNA and ssRNA are remarkably similar and dominated by π-stacking interactions with exposed aromatic residues (Figure 6B). Consistent with solution binding experiments and bacterial Csp:ssDNA/RNA structures [79,90], Lin28 CSD binds up to 8 nucleotides arranged in a curved single strand with defined orientation. In the case of the mLin28a:preE- let-7 structures, an additional ninth nucleotide is visible that establishes hydrogen bonds with the first base, thereby closing the preE stem loop.…”

Single-Stranded DNA-Binding Proteins: Multiple Domains for Multiple Functions

“…One of the OB folds from S. pombe Pot1 also discriminates >200-fold against RNA, but does so via steric exclusion of the 2′ hydroxyl as well as selective recognition of the 5-methyl group on one of the thymine bases (Lei et al, 2003). A similar discrimination between thymine and uracil is seen in the B. subtilis CspB protein (Sachs et al, 2012). Thus, a variety of mechanisms can be used, often at just a few key positions, to discriminate against RNA.…”

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins

“…To investigate this, we carried out all‐atom explicit solvent simulations of aggregating proteins, in their apo states. For this we used the following crystallographic structures of proteins for which experimental data on their APRs are known: PDB ids and their resolution‐ 1a1nB [2 Å], 1bebA[1.8 Å], 1hkqA [2.75 Å], 1ie4A [2.4 Å], 1kcqA [1.65 Å], 1wlaA [1.7 Å], 3pf4A [1.38 Å]. These proteins were chosen based on high resolution and few missing atoms in the structure.…”