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...

Sequence-specific 'H and '-N resonance asignments have been determined for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments. By use of these asgments, five antp lel 1-strands were identified from analysis of NMR data. Strands 1-4 have a clsical 3-2-1-4 Greek key -sheet topology and there are two 1-bulges, at positions Lys'0-Trp" and Gly"5-Asn". Threedimensional structures of CspA were generated from NMR data by using smulated annling with molecular dynamics. The overall chain fold of CspA Is a 1-barrel structure, with a tightly packed hydrophobic core. Two-dimensional isotopeedited pulsed-field gradient 15N-1H heteronuclear singlequantum coherence spectroscopy was used to chracerie the "N-1H fingerprint spectrum with and without a 24-base oligodeoxyribonucleotide, 5'-AACGGTTTGACGTACAGAC-CATTA-3'. Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule. This portion of the CspA molecular surface includes two putative RNA-binding sequence motifs which contribute to an unusual cluster of eight surface aromatic side chais: Trpol Phel", Phe", P , Phe3, His33, Phe3, and Tyr42. These surface aromatic groups, and also residues Lys"6, Ser", and Lys"' located on this same face of CspA, are higly conserved in the family of CspA homologues. These isotopeedited pulsed-field gradient NMR data provide a low-resolution mapping of a DNA-binding epitope on CspA.eukaryotic Y-box family of transcription-regulating proteins (6)(7)(8) (4,18), and these also contain RNP1 and RNP2 sequence motifs. The three-dimensional structure of one of these, the major cold shock protein CspB from Bacillus subtilis, has been determined recently by solution NMR (19) and x-ray crystallography (16). CspB has structural homology with several nucleic acid-binding proteins and forms complexes with ssDNA oligomers containing a CCAAT site in gel retardation experiments (16,19). In this paper we describe the overall chain fold of E. coli CspA determined in solution by NMR spectroscopy and demonstrate that CspA forms a complex with a 24-base oligodeoxyribonucleotide corresponding in sequence to the 5' leader region of cspA mRNA. 11 Many bacteria respond to low temperature stress by production ofcold shock proteins (1-5). In Escherichia coli this cold shock response involves enhanced production of at least 14 proteins (1, 2). One of these, the major cold shock protein (CspA), is not detectable when cells are grown at 370C but is produced at levels of 10-15% of total protein synthesis when cells are shifted to 10-15TC (2), with expression peaking 60-120 min following cold shock. The gene for CspA (cspA) has been cloned and sequenced (2), and primer extension studies indicate that levels of cspA transcripts also increase and then decrease in the cold shock response, paralleling the production kinetics of CspA protein. The transient nature of cold shock-induced cspA expression indicates a regulator...

We present grazing-incidence Fourier transform infrared and AFM data of Au, Al, and Ti vapor-deposited onto self-assembled monolayers (SAMs) of conjugated mono- and dithiols. SAMs of 4,4'''-dimercapto-p-quaterphenyl, 4,4"-dimercapto-p-terphenyl, and 4,4'-dimercapto-p-biphenyl have reactive thiols at the SAM/vacuum interface that interact with vapor-deposited Au or Al atoms, preventing metal penetration. Conjugated monothiols lack such metal blocking groups, and metals (Au, Al) can penetrate into their SAMs. Vapor deposition of Ti onto conjugated mono- and dithiol SAMs and onto hexadecanethiol SAMs destroys the monolayers.

SummaryThe gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine-Dalgarno sequence in the 159-base 5Ј-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37ЊC. This stabilization was found to be at least partially due to resistance against RNase E degradation. The coldshock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock lpp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37ЊC. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37ЊC. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37ЊC is prevented by destabilizing its mRNA.

We report a comprehensive study of the in-plane transport properties of Nd2 "Ce Cu04 & epitaxial thin films and crystals by both increasing and decreasing 6 with Ce content fixed at x = 0.15. We find a remarkable correlation between the appearance of superconductivity and (1) a positive magnetoresistance in the normal state, (2) a positive contribution to the otherwise negative Hall coefficient, and (3) an anomalously large Nernst effect. These results strongly suggest that both holes and electrons participate in the charge transport for the superconducting phase of Nd2, Ce"Cu04~. PACS numbers: 74.76.Bz, 72.15.Eb, 72.15.Gd, 74.25.Fy In most high-T, cuprates, such as La2, Sr Cu04 and YBa2Cu307, the charge carriers are doped holes. On the other hand, in Nd2 "Ce,Cu04 s (NCCO), where superconductivity is induced by substituting Nd3+ with Ce4+, the Cu02 planes are believed to be doped with electrons [1]. The "electron-doped" character of NCCO gives a strong constraint on the possible mechanisms for hightemperature superconductivity in copper oxides [2,3]. Recently this system has attracted much more interest because of its possible simple BCS s-wave pairing in the superconducting state [4], in contrast to d-wave behavior proposed for hole doped high-T, cuprates [5]. However, questions still remain concerning the nature of the charge carriers in the superconducting phase of NCCO. For instance, both