CspA, the Major Cold-shock Protein of Escherichia coli, Is an RNA Chaperone

Abstract

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.

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

“…in the repression of induction of CspA, CspB and CspG after acclimatization to cold shock . CspA has recently been shown to bind to RNA in vitro (Jiang et al, 1997). In this report, CspB, CspC and CspD from B. subtilis are shown to bind to RNA with moderately lower affinity than to the corresponding ssDNA.…”

“…According to this finding, a minimal concentration of CspB for binding to 54mer RNA is 5 × 10 ¹6 M, which suggests that in vivo mRNA could be bound even more efficiently than the oligonucleotides used in our assays. By analogy, a minimal concentration of CspA from E. coli was shown to be necessary for co-operative binding to RNA in vitro (Jiang et al, 1997). 5Ј UTRs of cspB and cspC revealed a highly conserved motif (CS-box 1) at their 5Ј end, which could be a target for regulation of translation.…”

p53-dependent induction of WAF1 by cold shock in human glioblastoma cells

“…Cold shock-induced proteins (Csps) have been well studied in Escherichia coli (Goldstein et al, 1990;Jones and Inouye, 1994;Jones et al, 1996). In contrast to hsps which are protein chaperones to help to refold the non-native proteins, prevent their aggregation or direct protein degradation (review in Becker and Craig, 1994), Csps have been shown to be RNA chaperones to prevent the formation of secondary structures in RNA at low temperature (Jones et al, 1996;Jiang et al, 1997). To our knowledge, cold shock proteins have not yet been found in human cells, thus, the e ects of cold shock on mammalian cells are poorly documented.…”

Conservation of the Cold Shock Domain Protein Family in Plants

“…CspA, the most prominent of the nine-member E. coli CSP family, accumulates up to 10% of total proteins during cold stress (Jiang et al, 1997). The three-dimensional structure of E. coli CspA forms a five-stranded ␤-barrel structure (Newkirk et al, 1994;Schindelin et al, 1994) that contains two consensus RNA-binding motifs (RNP1 and RNP2), which facilitate nucleic acid recognition/binding (Schroder et al, 1995).…”

“…The three-dimensional structure of E. coli CspA forms a five-stranded ␤-barrel structure (Newkirk et al, 1994;Schindelin et al, 1994) that contains two consensus RNA-binding motifs (RNP1 and RNP2), which facilitate nucleic acid recognition/binding (Schroder et al, 1995). CspA has been hypothesized to prevent RNA secondary structure formation (Jiang et al, 1997), thereby enhancing translation at low temperature.…”