CspA, the major cold-shock protein of Escherichia coli, is dramatically induced during the cold-shock response. The amino acid sequence of CspA shows 43% identity to the "cold-shock domain" of the eukaryotic Y-box protein family, which interacts with RNA and DNA to regulate their functions. Here, we demonstrate that CspA binds to RNA as a chaperone. First, CspA cooperatively binds to heat-denatured single-stranded RNA if it is larger than 74 bases, causing a supershift in gel electrophoresis. A minimal concentration of CspA at 2.7 ؋ 10 ؊5 M is absolutely required for this cooperative binding, which is sufficiently lower than the estimated cellular concentration of CspA (10 ؊4 M) in cold-shocked cells. No specific RNA sequences for CspA binding were identified, indicating that it has a broad sequence specificity for its binding. When the 142-base 5-untranslated region of the cspA mRNA was used as a substrate for ribonucleases A and T1, the addition of CspA significantly stimulated RNA hydrolysis by preventing the formation of RNase-resistant bands due to stable secondary structures in the 5-untranslated region. These results indicate that binding of CspA to RNA destabilizes RNA secondary structures to make them susceptible to ribonucleases. We propose that CspA functions as an RNA chaperone to prevent the formation of secondary structures in RNA molecules at low temperature. Such a function may be crucial for efficient translation of mRNAs at low temperatures and may also have an effect on transcription.

Lurcher (Lc) is a spontaneous, semidominant mouse neurological mutation. Heterozygous Lurcher mice (Lc/+) display ataxia as a result of a selective, cell-autonomous and apoptotic death of cerebellar Purkinje cells during postnatal development. Homozygous Lurcher mice (Lc/Lc) die shortly after birth because of a massive loss of mid- and hindbrain neurons during late embryogenesis. We have used positional cloning to identify the mutations responsible for neurodegeneration in two independent Lc alleles as G-to-A transitions that change a highly conserved alanine to a threonine residue in transmembrane domain III of the mouse delta2 glutamate receptor gene (GluR delta2). Lc/+ Purkinje cells have a very high membrane conductance and a depolarized resting potential, indicating the presence of a large, constitutive inward current. Expression of the mutant GluR delta2(Lc) protein in Xenopus oocytes confirmed these results, demonstrating that Lc is inherited as a neurodegenerative disorder resulting from a gain-of-function mutation in a glutamate receptor gene. Thus the activation of apoptotic neuronal death in Lurcher mice may provide a physiologically relevant model for excitotoxic cell death.

The major cold shock protein of Eschenchia coli, CspA, produced upon a rapid downshift in growth temperature, Is involved in the transcriptional regulation of at least two genes. The protein shares high homology with the nucleic acid-binding do of the Y-box factors, a family of eukaryotic proteins involved in nscriptional and trandational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA Is composed of five antiparallel -strands forming a osed five-stranded (-barrel. The three-dimensional structure of CspA Is similar to that of the major cold shock protein of BaciUlus subtils, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer Is formed in the crystal. The surface of CspA is characteristic for a protein interacting with single-stranded nucleic acds. Due to the high homology of the bacterial cold shock proteins with the Y-box factors, E. coft CspA and B. subdis CspB define a sructural framework for the common cold shock domain.The cold shock response inEscherichia colifollows an abrupt shift in growth temperature from 370C to 100C, inducing a lag phase in cell growth of 4-5 hr. It is accompanied by a severe reduction in protein synthesis (1). As a consequence of this cold shock, the relative rate of production of at least 14 cold shock proteins is increased. For 13 out of the 14 proteins the increase is 2-to 10-fold whereas synthesis of the major cold shock protein, CspA (CS 7.4), increases at least 100-fold, reaching a level of >10% oftotal protein synthesis at 100C (2). Other proteins expressed as part of the cold shock response of E. coli include NusA, RecA, polynucleotide phosphorylase, translation initiation factors 2a and 2p, pyruvate dehydrogenase (lipoamide), dihydrolipoamide acetyltransferase of pyruvate dehydrogenase, the nucleoid protein H-NS, and subunit A of DNA gyrase (1,3,4).CspA is a small hydrophilic protein consisting of 70 amino acids. It has striking similarity, at the level of 43% sequence identity (Fig. 1), with one domain ofthe Y-box factors, which is referred to as the cold shock domain (5, 6). The Y-box factors are a family of eukaryotic nucleic acid-binding proteins that preferentially bind to the Y box, an element of sequence CTAAIT-ClQYYAA found in the promoter regions of mammalian major histocompatibility complex class II genes (6). Within this sequence the underlined pentamer is especially conserved. Members of this family have also been found to bind to mRNA and to regulate translation in germ cells (7,8).CspA was shown to act as a transcriptional activator of the hns and gyrA genes encoding two other cold shock proteins (3,4). The promoter of hns contains one ATTGG element, whereas the promoter of gyrA contains three such elements, one of which is required for specific CspA-DNA interaction (4). ATTGG elements have been identified also in the promoter regions of genes encoding RecA, NusA, and polynucleotide phosphorylase, suggesting a common mechanism for induction...

A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15°C. The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein. Furthermore, after the shift from 37 to 15°C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15°C. The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA was found to unwind double-stranded RNA in the absence of ATP. Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA.Bacterial adaptation to various environmental stresses has been extensively investigated (reviewed in refs. 1-4). Interestingly, it has been demonstrated that Escherichia coli has an adaptive response not only to high temperature by inducing a group of heat-shock proteins but also to low temperature by inducing a group of cold-shock proteins (5, 6). In contrast to heat-shock proteins, which include protein chaperones required for protein folding and peptidases, cold-shock proteins appear to be involved in various cellular functions such as transcription, translation, and DNA recombination (5, 6).Among the cold-shock proteins of E. coli, CspA has been identified as the major cold-shock protein, which is almost exclusively produced at low temperature at a level of 250,000 molecules per cell (5, 7). The three-dimensional structure of CspA consisting of 69 amino acid residues has been determined, which is composed of five antiparallel (3-sheet structures (8,9). CspA binds to single-stranded DNA (8), and its possible function as an RNA chaperone has been speculated (6). In addition to CspA, E. coli contains a large family of CspA-like proteins consisting of CspB, CspC, CspD, and CspE, among which only CspB is a cold-shock protein (10, 11).In the present paper, we report a newly discovered coldshock protein of 70 kDa, which is also almost exclusively produced upon a temperature shift from 37 to 15°C, similar to the induction of CspA. It was found that this newly identified cold-shock protein is exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein at low temperature. This protein was purified and identified to be the product of the gene that has been known as deaD. This gene had been isolated as a multicopy suppressor for a temperature-sensitive mutation located in the gene encoding ribosomal S2 protein and proposed to encode a putative ATP-dependent RNA helicase based on sequence similarities with other known DEAD-box proteins (12). This protein now is assigned CsdA for cold-shock DEAD-box protein A. We found that this protein has a helix-destabilizing activity. Disruption of the gene resulted in a defect in growth a...