Biomedical subjects

I Grummt

Publications and source records attributed to I Grummt.

At least 55 records · Page 3Linked to original sources

PubMed1996

Species specificity of ribosomal gene transcription: a factor associated with human RNA polymerase I prevents transcription of mouse rDNA.

An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF-IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species-specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB-directed initiation complexes at the murine rDNA promoter.

Animals↗

PubMed1995

Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription factor UBF.

The nucleolar factor UBF is phosphorylated by casein kinase II (CKII) at serine residues within the C-terminal acidic domain which is required for transcription activation. To investigate the biological significance of UBF modification, we have compared the trans-activating properties of cellular UBF and recombinant UBF expressed in Escherichia coli. Using a variety of assays we demonstrate that unphosphorylated UBF is transcriptionally inactive and has to be phosphorylated at multiple sites to stimulate transcription. Examination of cDNA mutants in which the serine residues within the C-terminal domain were altered by site-directed mutagenesis demonstrates that CKII-mediated phosphorylations of UBF contribute to, but are not sufficient for, transcriptional activation. Besides CKII, other cellular protein kinases phosphorylate UBF at distinct sites in a growth-dependent manner. The marked differences in the tryptic peptide maps of UBF from growing and serum-starved cells suggest that alterations in the degree of UBF phosphorylation may modulate rRNA synthetic activity in response to extracellular signals.

3T3 Cells↗

PubMed1995

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription.

Amino Acid Sequence↗

PubMed1995

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.

3T3 Cells↗

PubMed1995

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of approximately 350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.

Adenosine Triphosphate↗

PubMed1995

Species specificity of transcription by RNA polymerase I.

An unusual property of ribosomal gene transcription is its marked species specificity. This results from distinct promoter-recognition properties of the RNA polymerase I transcription apparatus. The purification and functional characterization of TIF-IB/SL1, a promoter-recognition factor containing the TATA-binding protein, as well as the recent cloning of cDNAs encoding the three subunits (TAF(I)s) of the respective human and mouse factor, will facilitate the molecular analysis of the mechanisms underlying species-specific rDNA transcription and reveal how the basal transcriptional machinery has evolved.

Animals↗

PubMed1994

Yeast TBP can replace its human homologue in the RNA polymerase I-specific multisubunit factor SL1.

Basic mechanisms of transcription initiation are conserved from yeast to man. However, in contrast to genes transcribed by RNA polymerases II and III, ribosomal gene transcription by RNA polymerase I (Pol I) is species-specific. Promoter selectivity is mediated by SL1/TIF-IB, a multiprotein complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) which bind to DNA and nucleate the assembly of initiation complexes. Using a human cell line that expresses epitope-tagged yeast TBP, we have isolated a chimeric complex consisting of yeast TBP and human TAFs which faithfully promotes human rDNA transcription in vitro. This result argues that specific interactions between TBP and Pol I-specific TAFs have been evolutionarily conserved between distant species. In addition, this finding also underscores the importance of TAFs in determining promoter selectivity of Pol I.

Blotting, Western↗

PubMed1994

TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I.

We have characterized a transcription factor from Ehrlich ascites cells that is required for ribosomal gene transcription by RNA polymerase I (Pol I). This factor, termed TIF-IC, has a native molecular mass of 65 kDa, associates with Pol I, and is required both for the assembly of Sarkosyl-resistant initiation complexes and for the formation of the first internucleotide bonds. In addition to its function in transcription initiation, TIF-IC also plays a role in elongation of nascent RNA chains. At suboptimal levels of TIF-IC, transcripts with heterogeneous 3' ends are formed which are chased into full-length transcripts by the addition of more TIF-IC. Moreover, on a tailed template, which allows initiation in the absence of auxiliary factors, TIF-IC was found to stimulate the overall rate of transcription elongation and suppress pausing of Pol I. Thus TIF-IC appears to serve a function similar to the Pol II-specific factor TFIIF which is required for Pol II transcription initiation and elongation.

Animals↗

PubMed1994

The conserved core domain of the human TATA binding protein is sufficient to assemble the multisubunit RNA polymerase I-specific transcription factor SL1.

The human ribosomal RNA polymerase (Pol) I promoter selectivity factor SL1 is a complex consisting of the TATA binding protein (TBP) and three TBP-associated factors (TAFs). We have investigated which elements of TBP are involved in the assembly of Pol I-specific TBP-TAF complexes by comparing SL1 isolated from two human cell lines, one expressing epitope-tagged full-length TBP and another expressing a deletion of nearly the entire N-terminal domain (e delta NTBP). We have immunopurified epitope-tagged full-length TBP- and e delta NTBP-TAF complexes and show that e delta NTBP reconstitutes SL1 activity almost as well as full-length TBP. Moreover, e delta NTBP is shown to be associated with all three Pol I-specific TAFs. Thus, the core of TBP alone is sufficient for the correct assembly of the Pol I-specific TBP-TAF complex, and the variable N-terminal region of human TBP is not required for transcriptional activity. We also demonstrate by an in vitro protein-protein interaction assay that TBP directly interacts with the smallest TAF, TAFI48.

Amino Acid Sequence↗

PubMed1994

TBP-associated factors interact with DNA and govern species specificity of RNA polymerase I transcription.

Unlike genes transcribed by RNA polymerases II and III, transcription by RNA polymerase I is highly species-specific. Ribosomal promoter selectivity is brought about by a multisubunit transcription factor (SL1/TIF-IB) which consists of the TATA-binding protein (TBP) and three TBP-associated factors (TAFs). To determine the basis for the inability of SL1/TIF-IB to recognize heterologous rDNA, the transcriptional properties and the subunit composition of the murine and the human factor, as well as a chimeric complex containing epitope-tagged human TBP and murine TAFs, have been compared. We show that TBP can be exchanged between the human and mouse factor indicating that the variable N-terminal domain of TBP does not play a significant role in rDNA promoter selectivity. Instead, DNA binding is brought about by the TAFs. UV crosslinking experiments demonstrate that binding to the ribosomal gene promoter is mediated by two TAFs (TAFI48 and TAFI68) which have the same electrophoretic mobility in the human and mouse factor. The largest TAF is different in both species and is suggested to play a role in the species-specific assembly of productive preinitiation complexes. Thus, evolutionary changes of rDNA promoter sequences have been accompanied by changes in specific TAFs.

Amino Acid Sequence↗

PubMed1994

The N-terminal domain of the human TATA-binding protein plays a role in transcription from TATA-containing RNA polymerase II and III promoters.

In eukaryotes, the TATA box binding protein (TBP) is an integral component of the transcription initiation complexes of all three classes of nuclear RNA polymerases. In this study we have investigated the role of the N-terminal region of human TBP in transcription initiation from RNA polymerase (Pol) I, II and III promoters by using three monoclonal antibodies (mAbs). Each antibody recognizes a distinct epitope in the N-terminal domain of human TBP. We demonstrate that these antibodies differentially affect transcription from distinct classes of promoters. One antibody, mAb1C2, and a synthetic peptide comprising its epitope selectively inhibited in vitro transcription from TATA-containing, but not from TATA-less promoters, irrespective of whether they were transcribed by Pol II or Pol III. Transcription by Pol I, on the other hand, was not affected. Two other antibodies and their respective epitope peptides did not affect transcription from any of the promoters tested. Order of addition experiments indicate that mAb1C2 did not prevent binding of TBP to the TATA box or the formation of the TBP-TFIIA-TFIIB complex but rather inhibited a subsequent step of preinitiation complex formation. These data suggest that a defined region within the N-terminal domain of human TBP may be involved in specific protein-protein interactions required for the assembly of functional preinitiation complexes on TATA-containing, but not on TATA-less promoters.

Antibodies, Monoclonal↗

PubMed1994

Functional differences between the two splice variants of the nucleolar transcription factor UBF: the second HMG box determines specificity of DNA binding and transcriptional activity.

The nucleolar transcription factor UBF consists of two proteins, UBF1 and UBF2, which originate by alternative splicing. Here we show that deletion of 37 amino acids within the second of five HMG box motifs in UBF2 is important for the dual role of UBF as transcriptional activator and antirepressor. UBF1 is a potent antirepressor and transcriptional activator, whereas the ability of UBF2 to counteract histone H1-mediated repression and to stimulate ribosomal gene transcription both in vivo and in vitro is at least one order of magnitude lower. The difference in transcriptional activity between UBF1 and UBF2 is due to their different binding to the ribosomal gene promoter and enhancer. Apparently, the presence of an intact HMG box2 modulates the sequence-specific binding of UBF to rDNA control elements. However, the interaction of UBF with rDNA does not entirely depend on sequence recognition. Both UBF isoforms bind efficiently to four-way junction DNA, indicating that they recognize defined DNA structures rather than specific sequences. The results demonstrate that the HMG boxes are functionally diverse and that HMG box2 plays an important role in specific binding of UBF to rDNA.

3T3 Cells↗

PubMed1994

The HMG box-containing nucleolar transcription factor UBF interacts with a specific subunit of RNA polymerase I.

The mammalian transcription activator protein UBF contains five tandemly repeated HMG homology domains which are required for DNA binding. We have used highly purified RNA polymerase I (Pol I) and upstream binding factor (UBF) and investigated whether these two proteins interact in solution. We show by a variety of different experimental approaches, such as immunoprecipitation, glycerol gradient sedimentation, affinity chromatography and protein blotting, that UBF physically associates with Pol I. Mutational analysis reveals that the HMG boxes play an important role in this specific interaction. UBF binds to mouse and yeast Pol I, demonstrating that the interaction of UBF with Pol I has been conserved during evolution. Interestingly, in both species one Pol I-specific subunit (34.5 kDa in yeast and 62 kDa in mouse) was recognized by UBF. No specific interaction was observed with Pol II. Unexpectedly, UBF was found to associate also with a unique subunit of yeast Pol III. This apparent specific interaction of UBF with the two classes of RNA polymerases may reflect functionally important interactions of HMG box-containing transcription factors with the transcriptional apparatus.

Animals↗

PubMed1993

A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I.

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity.

Animals↗

PubMed1993

Sequence-specific transactivators counteract topoisomerase II-mediated inhibition of in vitro transcription by RNA polymerases I and II.

An inhibitor of RNA polymerase II transcription in vitro has been purified from HeLa cell nuclear extracts. Partial amino acid sequences derived from the purified protein revealed that the inhibitor of transcription corresponded to human topoisomerase II. Order of addition experiments provided evidence indicating that topoisomerase II inhibited transcription by binding over the core promoter and blocking preinitiation complex formation. Topoisomerase II-mediated repression could be relieved by sequence-specific transcriptional activators, having different activating and/or DNA binding domains, but antirepression required a transcriptional activation function in addition to a DNA binding domain. Moreover, transcription by RNA polymerase I was also inhibited by topoisomerase II and this inhibition could be relieved by the RNA polymerase I transactivator UBF. These observations suggest that topoisomerase II may participate in a general repression of transcription which can be counteracted by transcriptional activators.

Amino Acid Sequence↗

PubMed1993

The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription.

Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1). We have purified and functionally characterized this repressor protein from Ehrlich ascites cells. The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively. Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments. The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting. The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases. Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition. The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis.

Animals↗

PubMed1993

The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes.

We have characterized an anti-NOR (nucleolar organizer region) serum (P419) from a patient with rheumatoid arthritis and show that it contains antibodies directed against the RNA polymerase I-specific transcription initiation factor UBF. This serum reacts with UBF from a variety of vertebrate cells as revealed both by immunoblotting and by indirect immunofluorescence. We have used the P419 serum to study the intracellular localization of this transcription factor at the light and electron microscopic level. In interphase cells, UBF exhibits a pronounced punctate pattern and is found to be associated with necklace-like structures, which appear to reflect the transcriptionally active state of the nucleolus. Inhibition of rRNA synthetic activity caused either by nutritional starvation or by actinomycin D treatment resulted in a marked decrease in the number and in a significant increase in the size of UBF-positive granules. Under all experimental conditions applied, UBF was exclusively found within the nucleolus and was not released into the nucleoplasm or cytoplasm. During mitosis, UBF was found to be concentrated at the chromosomal NOR indicating that a significant quantity, if not all, of this factor remains bound to the ribosomal transcription units. From this we conclude that UBF is associated both with transcriptionally active and inactive rRNA genes and, therefore, changes in the intracellular localization of UBF are very likely not involved in rDNA transcription regulation.

Animals↗

PubMed1993

Function of the growth-regulated transcription initiation factor TIF-IA in initiation complex formation at the murine ribosomal gene promoter.

Alterations in the rate of cell proliferation are accompanied by changes in the transcription of rRNA genes. In mammals, this growth-dependent regulation of transcription of genes coding for rRNA (rDNA) is due to reduction of the amount or activity of an essential transcription factor, called TIF-IA. Extracts prepared from quiescent cells lack this factor activity and, therefore, are transcriptionally inactive. We have purified TIF-IA from exponentially growing cells and have shown that it is a polypeptide with a molecular mass of 75 kDa which exists as a monomer in solution. Using a reconstituted transcription system consisting of purified transcription factors, we demonstrate that TIF-IA is a bona fide transcription initiation factor which interacts with RNA polymerase I. Preinitiation complexes can be assembled in the absence of TIF-IA, but formation of the first phosphodiester bonds of nascent rRNA is precluded. After initiation, TIF-IA is liberated from the initiation complex and facilitates transcription from templates bearing preinitiation complexes which lack TIF-IA. Despite the pronounced species specificity of class I gene transcription, this growth-dependent factor has been identified not only in mouse but also in human cells. Murine TIF-IA complements extracts from both growth-inhibited mouse and human cells. The analogous human activity appears to be similar or identical to that of TIF-IA. Therefore, despite the fact that the RNA polymerase transcription system has evolved sufficiently rapidly that an rDNA promoter from one species will not function in another species, the basic mechanisms that adapt ribosome synthesis to cell proliferation have been conserved.

Animals↗