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I Grummt

Publications and source records attributed to I Grummt.

At least 37 records · Page 2Linked to original sources
PubMed1998

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

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.

Amino Acid Sequence↗
PubMed1998

Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors.

Transcription initiation of ribosomal RNA genes requires RNA polymerase I (Pol I) and auxiliary factors which either bind directly to the rDNA promoter, e.g. TIF-IB/SL1 and UBF, or are assembled into productive transcription initiation complexes via interaction with Pol I, e.g. TIF-IA, and TIF-IC. Here we show that all components required for specific rDNA transcription initiation are capable of physical interaction with Pol I in the absence of DNA and can be co-immunoprecipitated with antibodies against defined subunits of murine Pol I. Sucrose gradient centrifugation and fractionation on gel filtration columns reveals that approximately 10% of cellular Pol I elutes as a defined complex with an apparent molecular mass of > 2000 kDa. The large Pol I complex contains saturating levels of TIF-IA, TIF-IB and UBF, but limiting amounts of TIF-IC. In support of the existence of a functional complex between Pol I and basal factors, the large complex is transcriptionally active after complementation with TIF-IC. The results suggest that, analogous to class II gene transcription, a pre-assembled complex, the "Pol I holoenzyme", exists that appears to be the initiation-competent form of Pol I.

Animals↗
PubMed1997

Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I.

A replication fork barrier (RFB) at the 3' end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3' terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I-specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

3T3 Cells↗
PubMed1997

RNA polymerase I transcription termination: similar mechanisms are employed by yeast and mammals.

Termination of RNA polymerase I (Pol I) transcription requires the interaction of a specific DNA binding factor with terminator elements downstream of the pre-rRNA coding region. Both the terminator elements and the respective termination factors are distinct in yeast and mammals, and differences in the mechanism of transcription termination have been postulated. We have compared in vitro transcription termination of yeast and mouse Pol I using both the murine factor TTF-I, and the yeast homolog Reb1p. We show that, similar to TTF-I, Reb1p was sufficient for pausing of Pol I from either species, but was unable to cause release of the nascent transcripts from the paused ternary complex. The deficiency of Reb1p to mediate transcript release from Pol I of either species was complemented by the recently characterized murine release factor. Thus, both yeast and mouse Pol I termination requires a trans-acting factor that, in conjunction with the T-rich flanking sequence, releases the transcripts and Pol I from the template. The observation that the murine factor causes dissociation of ternary transcription complexes arrested by Reb1p suggests that the mechanism of Pol I termination is highly conserved from yeast to mammals.

Animals↗
PubMed1997

High mobility group 1 protein is not stably associated with the chromosomes of somatic cells.

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

3T3 Cells↗
PubMed1997

Oligomerization of the transcription termination factor TTF-I: implications for the structural organization of ribosomal transcription units.

Mammalian ribosomal genes are flanked at their 5'and 3'ends by terminator sequences which are recognized by the transcription termination factor TTF-I. The occurrence of the same binding site upstream and downstream of the gene raises the possibility that TTF-I can interact with both sequences simultaneously and thus brings the terminator in the vicinity of the gene promoter by looping out the pre-rRNA coding sequence. To test this model, we have examined the ability of TTF-I to oligomerize and found that both full-length and N-terminally truncated versions of TTF-I form stable oligomeric structures. At least two domains of TTF-I located within the 184 N-terminal and 445 C-terminal amino acids, respectively, mediate the self-association of several TTF-I molecules. In support of the looping model, TTF-I is capable of linking two separate DNA fragments via binding to the target sites. This result indicates that in addition to its function in transcription termination, TTF-I may serve a role in the structural organization of the ribosomal genes which may be important for maintaining the high loading density of RNA polymerase I on active rRNA genes.

Animals↗
PubMed1997

Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1.

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.

Amino Acid Sequence↗
PubMed1997

RNA polymerase I transcription on nucleosomal templates: the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression.

Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator.

Animals↗
PubMed1997

Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer.

We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulatory elements. Hypersensitive sites map to the transcription initiation site, the enhancer repeats, the spacer promoter and two sequence elements which coincide with amplification-promoting sequences. Analysis of the DNA curvature by computer modeling uncovered a striking correlation between sequence-directed structural features of regulatory regions and the position of nuclease hypersensitive sites. Moreover, we demonstrate that nucleosomes are specifically positioned upstream and downstream of the transcription start site. In vitro studies using chromatin assembled in the presence of Drosophila embryo extracts show that binding of the transcription termination factor TTF-I to the upstream terminator mediates this specific nucleosome positioning at the rDNA promoter in an ATP- dependent fashion.

3T3 Cells↗
PubMed1997

Identification of a transcript release activity acting on ternary transcription complexes containing murine RNA polymerase I.

Termination of mammalian ribosomal gene transcription by RNA polymerase I (Pol I) requires binding of the nucleolar factor TTF-I (transcription termination factor for Pol I) to specific rDNA terminator elements. We have used recombinant murine TTF-I in an immobilized tailed template assay to analyze individual steps of the termination reaction. We demonstrate that, besides the TTF-I-DNA complex which stops elongating Pol I, an additional activity is required to release both the nascent transcript and Pol I from the template. Moreover, transcript release, but not TTF-I-directed pausing, depends on upstream sequences directly flanking the terminator element. Together, complete termination of Pol I transcription requires TTF-I bound to the terminator DNA, a stretch of thymidine residues upstream of the TTF-I-mediated pause site and an activity which releases the RNA transcript and Pol I from the DNA template.

Animals↗
PubMed1997

Constitutive and strong association of PAF53 with RNA polymerase I.

Mouse RNA polymerase I (Pol I) is composed of 14 polypeptides, 3 of which are thought to be loosely associated with, and may be dislodged from, Pol I. To find out whether these polymerase-associated factors (PAF53, 51, and 49) serve a role in growth-dependent control of rDNA transcription, we generated polyclonal antibodies against three subunits of murine Pol I, RPA116, RPA40 and PAF53, and used different experimental approaches, e.g. immunoblot analysis, immunoprecipitation and immunofluorescence studies, to compare the stoichiometry of individual subunits both in different Pol I preparations and in extracts from cells grown under different conditions. This comparative analysis reveals that the molar ratio of the second largest subunit RPA116 to PAF53 is the same, irrespective of whether crude extracts or highly purified Pol I fractions are analyzed. Significantly, the relative level of PAF53 was comparable in exponentially growing or growth-arrested cells, indicating that growth-dependent fluctuations in Pol I activity are not accompanied by alterations in the amount of PAF53. In addition, we show by high resolution immunofluorescence analysis that, under conditions of repressed rDNA transcription, including serum starvation, actinomycin treatment und during mitosis, PAF53 remains attached to the transcriptional machinery. The finding that the Mr 53,000 protein remains in the multiprotein complex under all experimental conditions tested indicates that PAF53 is not a loosely associated regulatory factor but a bona fide subunit of Pol I.

3T3 Cells↗
PubMed1997

Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA polymerase I.

We describe the cloning and analysis of mRPA1, the cDNA encoding the largest subunit (RPA194) of murine RNA polymerase I. The coding region comprises an open reading frame of 5151 bp that encodes a polypeptide of 1717 amino acids with a calculated molecular mass of 194 kDa. Alignment of the deduced protein sequence reveals homology to the beta' subunit of Escherichia coli RNA polymerase in the conserved regions a-h present in all large subunits of RNA polymerases. However, the overall sequence homology among the conserved regions of RPA1 from different species is significantly lower than that observed in the corresponding beta'-like subunits of class II and III RNA polymerase. We have raised two types of antibodies which are directed against the conserved regions c and f of RPA194. Both antibodies are monospecific for RPA194 and do not cross-react with subunits of RNA polymerase II or III. Moreover, these antibodies immunoprecipitate RNA polymerase I both from murine and human cell extracts and, therefore, represent an invaluable tool for the identification of RNA polymerase I-associated proteins.

Amino Acid Sequence↗
PubMed1997

Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein.

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.

Animals↗
PubMed1996

Molecular cloning of RPA2, the gene encoding the second largest subunit of mouse RNA polymerase I.

We have cloned the cDNA encoding the second largest subunit of RNA polymerase I, termed RPA2, from mouse cells. The cDNA has a 3978-nucleotide open reading frame encoding a polypeptide of 1136 amino acids with a calculated molecular mass of 128 kDa. A sequence alignment of mouse RPA2 with the corresponding gene from Drosophila melanogaster and yeast reveals a much lower sequence similarity of this subunit of RNA polymerase I (Pol I) compared to the second largest subunit of other eukaryotic RNA polymerases. Four Pol I-specific regions, termed I alpha-I delta, are conserved in the N-terminal part of RPA2. The structural features of the different domains as well as the homology to essential functional domains found in other RNA polymerases are discussed.

Amino Acid Sequence↗
PubMed1996

The amino-terminal domain of the transcription termination factor TTF-I causes protein oligomerization and inhibition of DNA binding.

The transcription termination factor TTF-I binds specifically to an 18 bp DNA element in the murine ribosomal gene spacer and mediates termination of RNA polymerase I transcription. In this study, we have compared DNA binding and termination activity of recombinant full-length TTF-I (TTF-Ip130) with two deletion mutants lacking 184 and 322 N-terminal amino acids, respectively. All three proteins exhibit similar termination activity, but the DNA binding of TTF-Ip130 is at least one order of magnitude lower than that of the deletion mutants, indicating that the N-terminus represses the interaction of TTF-I with DNA. The inhibitory effect of the N-terminus can be transferred to a heterologous DNA binding domain and is separable from other activities of TTF-I. We show by several methods that TTF-Ip130, the N-terminal domain alone, and fusions of the N-terminus with the DNA binding domain of Oct2.2 form stable oligomers in solution. Thus, in contrast to previous studies suggesting that activation of TTF-I occurs through proteolysis, we demonstrate that full-length TTF-I mediates termination of rDNA transcription in vivo and in vitro and that the oligomerization state of TTF-I may influence its DNA binding activity.

Animals↗
PubMed1996

TFIIS binds to mouse RNA polymerase I and stimulates transcript elongation and hydrolytic cleavage of nascent rRNA.

Efficient transcription elongation by RNA polymerase I (Pol I) requires a specific Pol I-associated factor, termed TIF-IC. Here we show that TFIIS, a factor that has previously been shown to promote read-through past many types of blocks to elongation by RNA polymerase II, also enhances Pol I-directed transcription elongation. In a reconstituted transcription system containing purified proteins, TFIIS stimulates Pol I transcription by increasing the overall rate of RNA chain elongation. As with Pol II, ternary Pol I complexes cleave the 3' end of the nascent transcripts in response to TFIIS. The truncated RNAs remain bound to the template, are subject to pyrophosphorolysis, and can be chased into longer transcripts. Moreover, we show by immunoprecipitation and specific affinity chromatography that TFIIS physically interacts with Pol I. The results suggest that nascent transcript cleavage by TFIIS or a TFIIS-related factor may be a general mechanism by which both Pol I and Pol II can bypass transcriptional impediments.

Animals↗
PubMed1996

Mapping of replication initiation sites in the mouse ribosomal gene cluster.

We have used nascent strand determination analysis to map start sites of DNA replication in the mouse ribosomal gene cluster in which individual copies of the ribosomal genes are separated by intergenic spacer regions. One origin of bidirectional replication (OBR) was localized within a 3 kb region centered about 1.6 kb upstream of the rDNA transcription start site. At least one additional initiation site is situated near the 3' end of the transcription unit. Adjacent to the OBR at the transcription start site are located two amplification-promoting sequences, i.e., APS1 and APS2. Nuclease-hypersensitive sites were identified in both of the two APSs as well as in the OBR region, thus indicating that these sequences have an altered chromatin structure. In the OBR an intrinsically bent region, a purine-rich element and other prospective initiation zone components are found.

3T3 Cells↗