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Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes.

ABSTRACT: Termination of transcription by RNA polymerase I (Pol I) is a two-step process which involves pausing of elongating transcription complexes and release of both pre-rRNA and Pol I from the template. In mouse, pausing of elongation complexes is mediated by the transcription termination factor TTF-I bound to the 'Sal box' terminator downstream of the rDNA transcription unit. Dissociation of paused ternary complexes requires a cellular factor, termed PTRF for Pol I and transcript release factor. Here we describe the molecular cloning of a cDNA corresponding to murine PTRF. Recombinant PTRF is capable of dissociating ternary Pol I transcription complexes in vitro as revealed by release of both Pol I and nascent transcripts from the template. Consistent with its function in transcription termination, PTRF interacts with both TTF-I and Pol I. Moreover, we demonstrate specific binding of PTRF to transcripts containing the 3' end of pre-rRNA. Substitution of 3'-terminal uridylates by guanine residues abolishes PTRF binding and impairs release activity. The results reveal a network of protein-protein and protein-nucleic acid interactions that governs termination of Pol I transcription.

SUBMITTER: Jansa P

PROVIDER: S-EPMC1170626 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

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Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes.

Jansa P P Mason S W SW Hoffmann-Rohrer U U Grummt I I

The EMBO journal 19980501 10

Termination of transcription by RNA polymerase I (Pol I) is a two-step process which involves pausing of elongating transcription complexes and release of both pre-rRNA and Pol I from the template. In mouse, pausing of elongation complexes is mediated by the transcription termination factor TTF-I bound to the 'Sal box' terminator downstream of the rDNA transcription unit. Dissociation of paused ternary complexes requires a cellular factor, termed PTRF for Pol I and transcript release factor. Her ...[more]

PMID: 9582279

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Project description:BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5' cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ?26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix), except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski). Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA -transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p?=?0.0006). The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p?=?0.0001). Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal expression may be involved in the oncogenesis of human malignancies such as lung cancer.

Dataset Information

Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes.

Publications

Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes.

Similar Datasets

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