The major cold shock protein of Escheyichia coli, CS7.4, is produced at a level of 13% of total protein synthesis upon a temperature shift from 37 to 10'C. The transcription of its gene (cspA) was found to be tightly regulated and induced only at low temperature. In addition, the cspA mRNA was extremely unstable at 37'C, so that CS7.4 production was hardly detected when the culture temperature was shifted from 15'C to 37°C. The transcription initiation site (+1) was identified. In vivo footprinting demonstrated that the region from bases -35 to -73 was protected from chemical modification, and gel mobility shift analysis showed that a cold-shocked cell extract contained a factor(s) specifically bound to the fragment containing the sequence between bases -63 and -92. This factor was synthesized de novo only at low temperature, and its synthesis was inhibited by chloramphenicol. Possible functions of this factor are discussed.When exponentially growing cultures of Escherichia coli are transferred from 37°C to 10 or 15'C, the production of a 7.4-kDa cytoplasmic protein (CS7.4) is dramatically induced (8). The rate of production of CS7.4 reaches as high as 13% of total protein synthesis within 1 to 1.5 h after the temperature shift to 10°C. The gene for CS7.4 has been cloned; its nucleotide sequence indicated that CS7.4 consists of 70 amino acid residues. Interestingly, CS7.4 shows high sequence similarity with one of the domains of human DNAbinding proteins DbpA, DbpB, and YB-1 (6a, 27), raising the intriguing possibility that the conserved sequence in these proteins may play an essential role in gene regulation in both prokaryotes and eukaryotes.We demonstrated previously that the production of CS7.4 was very tightly regulated, such that CS7.4 synthesis was undetectable at 37°C (8). In this report, we demonstrate that the transcription of the gene for CS7.4 (cspA) dramatically increased upon cold shock. In addition, the cspA mRNA was extremely unstable at high temperature, which also contributes to the exclusive production of CS7.4 at low temperature. A distinct region upstream of the cspA promoter was protected in an in vivo footprinting experiment at low temperature. Subsequently, we were able to identify a factor(s) that bound to a specific region upstream of the cspA promoter. This factor was synthesized de novo only when cells were cold shocked. A possibility that this factor functions as a transcriptional factor for cspA is discussed. previously described (10, 24). Oligonucleotide-directed sitespecific mutagenesis was performed by the plasmid method as previously described (10). MATERIALS AND METHODSIsolation of RNA. RNA was isolated by the method of Chomczynski and Sacchi (3) with the following modifications. The denaturing solution consisted of 4 M guanidinium thiocyanate (Fluka), 2% sarcosyl, 50 mM Tris-HCl (pH 7.5), 12 mM EDTA, and 0.15 M 2-mercaptoethanol, and all solutions were treated with diethyl pyrocarbonate (Sigma) before they were autoclaved. A 50-ml cell culture was grown in L broth (15) at 37°C...

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SMRT (silencing mediator for retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor copressor) mediate transcriptional repression of important regulators that are involved in many signaling pathways. SMRT and N-CoR are related proteins that form complexes with mSin3A͞B and histone deacetylases to induce local chromatin condensation and transcriptional repression. However, SMRT is substantially smaller than N-CoR, lacking an N-terminal domain of approximately 1,000 aa that are present in N-CoR. Here, we report the identification of SMRTextended (SMRTe), which contains an N-terminal sequence that shows striking similarity with N-CoR. As in N-CoR, this SMRTe-N-terminal domain also represses basal transcription. We find that SMRTe expression is regulated during cell cycle progression and SMRTe transcripts are present in many embryonic tissues. These data redefine a structurally and functionally more related nuclear receptor corepressor family and suggest an additional role for SMRTe in the regulation of cycle-specific gene expression in diverse signaling pathways.Transcriptional repression plays an important role in the proper regulation of cell growth, differentiation, and development (1-3). In one mechanism of transcriptional inhibition, a repressor competes with an activator for DNA binding. Alternatively, transcriptional repressors also can inhibit basal transcription through direct interaction with general transcription factors, or indirectly by promoting chromatin condensation, thereby preventing the loading of general transcription factors to the promoter (1-3).Transcriptional repression by unliganded nuclear receptors such as TR (thyroid hormone receptor) and RAR (retinoic acid receptor) provide an excellent system for dissecting the pathways that lead to gene repression. TR and RAR play important roles in the regulation of cell growth, differentiation, and homeostasis. In the absence of hormone, TR and RAR actively repress target gene expression (4). Unliganded TR and RAR interact with the corepressors SMRT (silencing mediator for retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor corepressor) (5, 6), which are components of corepressor complexes that also contain mSin3A͞B and histone deacetylases (7-9). The binding of hormone to these receptors induces active conformations (10-12), causing dissociation of the corepressor complex. A coactivator complex then is recruited, leading to transcriptional activation (13).In addition to TR and RAR, SMRT and N-CoR interact with other transcriptional regulators involved in various signaling pathways. These regulators include the orphan nuclear receptors COUP-TF1 (14), Rev-Erb, RVR (15), and DAX-1 (16), the latter of which is involved in congenital X-linked adrenal hypoplasia (17). SMRT and N-CoR also interact with antagonist-bound progesterone and estrogen receptors (18,19), suggesting that the corepressors may play a role in determining the antagonistic activities of antihormones. Functional complexes containing SMRT and N-CoR al...