The Krfippel-associated box (KRAB) is a highly conserved, 75-aa region containing two predicted amphipathic a-helices. The KRAB domain is present in the amino-terminal regions of more than one-third of all Krfippelclass Cys2His2 zinc finger proteins and is conserved from yeast to man; however, its function is unknown.

A tetracycline-controlled transrepressor protein has been engineered to silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRAB domain of human Kox1 to the Tet repressor derived from Tn10 of Escherichia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells. The TetR-KRAB protein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline. When TetR-KRAB bound to tetO sequences upstream of the immediate-early promoter-enhancer of human cytomegalovirus (CMV), the expression of a CMV-driven luciferase reporter construct (ptetO7-CMV-L) was repressed in transient transfection experiments. This silencing was found to operate on different promoters and from tetO sequences placed more than 3 kb from the transcriptional start site. We constructed a stable, doubly transfected cell line (TIS-10) carrying a chromosomally integrated ptetO7-CMV-L reporter construct and expressing the TetR-KRAB protein. Inducible gene expression has been a valuable tool for the study of gene function in bacteria, yeasts, and Drosophila melanogaster. In mammalian cells, eukaryotic promoter systems that respond to inducing agents such as glucocorticoid hormone (19, 23), heat shock (40), heavy metal ions (28), or interferon (32) have been used. To circumvent limitations due either to the leakiness of utilized promoters or the pleiotropic effects of inducing agents, chimeric transcription factors have been generated, for example, by fusing the strong transcriptional activator domain of VP16 to the Tn10-derived prokaryotic tetracycline repressor (TetR) protein (16). In general, chimeric transcription factors of this kind, TetR-VP16 (16), GAL4-VP16 (33), LexA-VP16 (7), and LacR-VP16 (22), are targeted to minimal promoters which are fused to multiple DNA binding sites specific for the factor in question. This results in marked transcriptional stimulation of the respective minimal promoter. In order to modulate the transcriptional activity of chimeric transcription factors, movable steroid binding domains originating from the glucocorticoid receptor (30) or the estrogen receptor (38) have been fused to sequencespecific DNA binding domains. In the case of a GAL4-VP16-estrogen receptor fusion, estrogen administration has been shown to produce a functionally active chimeric transcription factor that activates a target promoter (6).In the tetracycline system, the TetR protein fused to the transactivating domain of VP16 (called tTA) has been shown to bind to and strongly activate minimal promoter systems containing seven tetracycline operator (tetO) sequences (15, 16). The binding of tTA to the tetO sequences is blocked by tetracycline, preventing the activation of the target promoter which generates a very tight genetic switch (16).In this report, we present a novel system for tetracyclinecontrolled silencing of eukaryotic promoters. Here, the KRAB repressor domain of the human ...

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The yeast PET1402/OXA1 gene encoding a 44.8-kDa protein is required for mitochondrial biogenesis. Substitution of Leu240 to serine in the protein results in an accumulation of the precursor form of the mitochondrially encoded subunit 2 of cytochrome oxidase (Cox2) and temperature-sensitive respiration. This temperature sensitivity can be suppressed by a mutation in the cox2 gene changing Ala189 of the Cox2 protein to proline. In the cox2-ts1402 double mutant respiration is restored without removal of the Cox2 pre-sequence. The suppression suggests an interaction of the Pet1402 protein with the cytochrome oxidase complex. Antibodies raised against the predicted C-terminus and the tagged N-terminus of the Pet1402 protein reacted with a 37-kDa polypeptide. This protein, present in the mitochondrial fraction, is localized within the inner membrane. The difference in size can be explained by the removal of the predicted mitochondrial-targeting sequence from the Pet1402 protein. The mitochondrial localization of the protein points to a direct interaction with the cytochrome oxidase complex.