Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar -sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. Similar to CspB, CspC and CspD unfold and refold extremely fast in a N º º U two-state reaction with average lifetimes of only 100 -150 ms for the native state and 1-6 ms for the unfolded states at 25°C. As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro. Analysis of the CSP stabilities in vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37°C as well as after cold shock. The cellular half-life of CspC is shortened at 37°C, but under cold shock conditions CspC becomes stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37°C and, in particular, under cold shock conditions.Cold shock proteins (CSPs) 1 are found in a wide range of Gram-positive and Gram-negative bacteria, often in families of three (as in Bacillus subtilis) to nine (as in Escherichia coli) highly homologous members (identity Ͼ70%) (for review see Ref. 1). Recently, CSPs were also found in Aquifex aeolicus (2) and Thermotoga maritima (3), indicating that CSPs were present at the origin of bacterial divergence and therefore are presumably an evolutionarily old class of proteins. A CSPhomologous domain (cold shock domain) is found in many eukaryotic nucleic acid-binding proteins (for review see Ref. 4), where it confers specific RNA binding (5, 6). CSPs bind to single-stranded DNA and RNA in a cooperative manner and with low sequence specificity (7-9). As a model for the cold shock domain, the structures of CspB (B. subtilis) and CspA (E. coli) were solved, revealing similar, compact five-stranded -barrel folds (10 -13). CSPs possess binding sites for singlestranded nucleic acids on their antiparallel three-stranded -sheets, which involve basic and aromatic residues. These are the so-called RNA-binding ribonucleoprotein motifs (13,14).CSPs were discovered originally because they are strongly induced in response to cold shock (15), and thus they were assumed to be important for adaptation to low temperatures. The major cold shock protein, CspA from E. coli, was in fact shown to increase the synthesis of several cold stress-inducible proteins after a decrease in temperature (16). However, different members of the E. coli CSP family are regulated differently and appear to perform functions also during cell division or during the stationary phase (17). Recent work shows that in B. subtilis, CSPs are essential for protein synthesis at low as well as at optimal temperature and also during the stationary phase (8). Moreover, CspA from E. coli destabilizes...