Biomedical subjects

I Grummt

Publications and source records attributed to I Grummt.

At least 73 records · Page 4Linked to original sources

PubMed1992

Limited proteolysis unmasks specific DNA-binding of the murine RNA polymerase I-specific transcription termination factor TTFI.

Previously we have shown that nuclear extracts from mouse cells contain a heterogeneous group of polypeptides (p65, p80, p90, p100) which form distinct DNA-protein complexes on the 18 base-pair sequence element (termed Sal-box), which constitutes the murine rDNA transcription termination signal. These distinct proteins mediate cessation of RNA polymerase I (pol I) transcription elongation and release of the nascent RNA chains, indicating that they function as termination factor(s). Here, we report the biochemical analysis of the pol I-specific transcription termination factor TTFI. We show that the heterogeneity of TTFI is due to limited proteolysis of a larger, 130 kDa precursor protein (p130). The DNA-binding activity of p130 is strongly reduced as compared to the proteolytic derivatives, indicating that the DNA-binding domain is repressed within the full-length molecule. We have used limited proteolysis to purify and functionally characterize a TTFI core polypeptide (p50) which still specifically binds to the Sal-box target sequence and directs rDNA transcription termination. The equilibrium constant of purified p50 to bind specifically to DNA is 9 x 10(9) M-1. Additionally, we demonstrate that TTFI binds to DNA as a monomer and that binding induces DNA bending. This observation suggests that not only specific DNA-protein and protein-protein interactions but also conformational alterations of DNA may play a role in the termination process.

Animals↗

PubMed1992

Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor.

In a reconstituted system consisting of partially purified RNA polymerase I (pol I) and the initiation factors TIF-IA, TIF-IB, and TIF-IC, the nucleolar factor UBF (upstream binding factor) stimulates transcription from the rRNA-encoding DNA (rDNA) promoter at least 50-fold. This activation is not observed at high template concentrations or in the presence of highly purified pol I. Template commitment experiments suggest that UBF activates transcription by relieving inhibition exerted by a negative-acting factor(s) in the polymerase fraction that competes for TIF-IB binding to the rDNA promoter and prevents the formation of preinitiation complexes. Using purified histone H1 bound to DNA as a model for the repressed state of the rDNA promoter, we show that UBF counteracts H1-mediated repression of pol I transcription. The implications of these findings are discussed with respect to the protein-protein and protein-DNA interactions at the rDNA promoter and the possible involvement of UBF in control of ribosomal gene transcription.

Animals↗

PubMed1992

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Amino Acid Sequence↗

PubMed1991

Transcription complex formation at the mouse rDNA promoter involves the stepwise association of four transcription factors and RNA polymerase I.

We have used purified transcription factors and RNA polymerase I (pol I) to analyze the individual steps involved in the formation of transcription initiation complexes at the mouse ribosomal gene promoter in vitro. Complete assembly of transcription complexes requires pol I and at least four auxiliary factors, termed TIF-IA, TIF-IB, TIF-IC, and UBF. Preincubation and template commitment, as well as order of addition protocols, were used to discriminate between various intermediate complexes generated during assembly of the initiation complex. As a first step, TIF-IB binds to the core promoter, a process that is facilitated by the upstream control element and the upstream binding factor (UBF). Binding of TIF-IB to the rDNA promoter results in the formation of a functional preinitiation complex (complex 1), which is stable for many rounds of transcription. UBF, which on its own does not stably associate with the rDNA promoter, triggers a 5-10-fold increase in the overall amount of this primary complex. Following binding of TIF-IB and UBF to the template DNA, pol I and TIF-IC successively bind, yielding complexes 2 and 3, respectively. Transcription-competent initiation complexes are built up by the final association of the growth-regulated factor TIF-IA. The various complexes can be distinguished by their different sensitivity to Sarkosyl. Only the complete complex consisting of all four factors and pol I shows resistance to intermediate concentrations of Sarkosyl (0.045%) and is competent to catalyze the formation of the first phosphodiester bond. The initiated complex is, on the other hand, resistant to high concentrations of Sarkosyl (0.3%). The hierarchical nature of the different complexes formed suggests a model for transcription initiation and predicts functions for the individual factors.

Animals↗

PubMed1991

Isolation of multiple protein factors involved in ribosomal DNA transcription.

Studies were made of the molecular mechanisms which regulate ribosomal gene transcription in response to changes in the growth rate of cells. Extracts prepared from exponentially growing Ehrlich ascites cells faithfully and efficiently transcribe cloned mouse rDNA, whereas extracts from growth-arrested cells are virtually inactive. In an attempt to identify and characterize functionally the proteins that mediate the accuracy and the control of transcription initiation, a fractionation procedure was developed which allows the purification of RNA polymerase I and four accessory factors that are required for transcription initiation at the ribosomal gene promoter. Starting from about 300 ml of cell extract, each of the individual factors and the polymerase was purified on at least four different chromatographic columns, including ion-exchange chromatography on DEAE-Sepharose, heparin-Ultrogel, Mono Q and Mono S, gel filtration and specific affinity chromatography. The resulting protein fractions are functionally active, as shown by reconstitution of specific rDNA transcription in the presence of purified polymerase and the additional factors.

Animals↗

PubMed1990

An undecamer DNA sequence directs termination of human ribosomal gene transcription.

Previously we have shown that a repetitive 18 bp sequence motif, the Sal box (AGGTCGACCAGA/TT/ANTCCG), present in the 3' terminal spacer of mouse rDNA constitutes a termination signal for RNA polymerase I (pol I). Similar sequence elements which are functionally analogous to the murine terminator are present in the spacer of human rDNA. However, the human termination signal is shorter encompassing only 11 bp (GGGTCGACCAG) which correspond to the proximal part of the mouse sequence. Two out of the five human Sal box elements are functionally inactive due to natural point mutations which damage factor binding. A similar sequence motif with a 10 of 11 base identity with the downstream terminators is located upstream of the human transcription initiation site. The upstream element interacts with the same factor(s) as the downstream terminators and is also capable to stop elongating human RNA polymerase I. Despite the human and mouse factors exert different electrophoretic mobilities in gel retardation assays, UV-crosslinking and proteolytic clipping experiments indicate that both the sizes and the tertiary structure of the Sal box binding proteins of both species are very similar. When bound to DNA, both the human and the mouse factor terminate transcription of pol I from the heterologous species. The results implicate that changes in signal sequences necessary for termination have been accompanied by compensatory changes in the DNA binding domain of the protein(s) interacting with the termination signal. In contrast, the protein-protein interactions between the termination factor and the transcribing RNA polymerase I appear to have been conserved during evolution.

Animals↗

PubMed1990

Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases.

The 18-base-pair sequence element AGGTCGACCAGTACTCCG (the Sal box) signals termination of mouse ribosomal gene transcription. This sequence is recognized by a sequence-specific DNA-binding protein, TTF I, which mediates the termination of transcription by RNA polymerase I (pol I). Subsequently, the ends of the primary transcripts are trimmed by 10 nucleotides in a sequence-dependent 3'-terminal processing reaction. We have now investigated whether TTF I bound to its target sequence will block elongation by any RNA polymerase by steric hindrance, or whether it is specific for elongation by pol I. The results demonstrate that TTF I directs transcription termination with RNA polymerase I from species as divergent as mouse and yeast, but fails to affect elongation by heterologous polymerases (eukaryotic RNA polymerases II and III, Escherichia coli or bacteriophage T3 RNA polymerase). By contrast, purified lac repressor bound to its operator sequence stops elongation by both RNA polymerase I and II.

Animals↗

PubMed1990

Isolation and functional characterization of TIF-IB, a factor that confers promoter specificity to mouse RNA polymerase I.

The murine ribosomal gene promoter contains two cis-acting control elements which operate in concert to promote efficient and accurate transcription initiation by RNA polymerase I. The start site proximal core element which is indispensable for promoter recognition by RNA polymerase I (pol I) encompasses sequences from position -39 to -1. An upstream control element (UCE) which is located between nucleotides -142 and -112 stimulates the efficiency of transcription initiation both in vivo and in vitro. Here we report the isolation and functional characterization of a specific rDNA binding protein, the transcription initiation factor TIF-IB, which specifically interacts with the core region of the mouse ribosomal RNA gene promoter. Highly purified TIF-IB complements transcriptional activity in the presence of two other essential initiation factors TIF-IA and TIF-IC. We demonstrate that the binding efficiency of purified TIF-IB to the core promoter is strongly enhanced by the presence in cis of the UCE. This positive effect of upstream sequences on TIF-IB binding is observed throughout the purification procedure suggesting that the synergistic action of the two distant promoter elements is not mediated by a protein different from TIF-IB. Increasing the distance between both control elements still facilitates stable factor binding but eliminates transcriptional activation. The results demonstrate that TIF-IB binding to the rDNA promoter is an essential early step in the assembly of a functional transcription initiation complex. The subsequent interaction of TIF-IB with other auxiliary transcription initiation factors, however, requires the correct spacing between the UCE and the core promoter element.

Animals↗

PubMed1990

A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis.

Control of mouse ribosomal RNA synthesis in response to extracellular signals is mediated by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. Factor TIF-IA interacts with RNA polymerase I (pol I), thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other auxiliary transcription initiation factors, designated TIF-IB, TIF-IC and UBF. With regard to several criteria, the growth-dependent factor TIF-IA behaves like a bacterial sigma factor: (i) it associates physically with pol I, (ii) it is required for initiation of transcription, (iii) it is present in limiting amounts and (iv) under certain salt conditions, it is chromatographically separable from the polymerase. In addition, evidence is presented that dephosphorylation of pol I abolishes in vitro transcription initiation from the ribosomal gene promoter without significantly affecting the polymerizing activity of the enzyme at nonspecific templates. The involvement of both a regulatory factor and post-translational modification of the transcribing enzyme provides an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to adapt ribosome synthesis rapidly to a variety of extracellular signals.

Animals↗

PubMed1990

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interaction with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription often fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.

Animals↗

PubMed1989

Transcriptional enhancement by upstream activators is brought about by different molecular mechanisms for class I and II RNA polymerase genes.

Ribosomal gene transcription requires the functional interplay of at least two promoter elements, the upstream control element (UCE) and the start site proximal core, which operate in concert to promote efficient and accurate transcription initiation by RNA polymerase I (pol I). Because this bipartite organization of the rDNA promoter is formally analogous to the organization of a typical pol II promoter, we have examined whether transcriptional activation by upstream activating sequences is brought about by similar molecular mechanisms for both classes of genes. We have replaced the UCE of the mouse rDNA promoter by three different pol II activating sequences (the yeast GAL4 binding sites, the target sequence of the enhancer binding protein E2 from bovine papilloma virus type 1 and the octamer motif), and measured the template activity of these chimeric promoters in the presence of the trans-activating proteins either in a cell free transcription system or in vivo after transfection into mouse cells. In the context of the pol I promoter none of these transcriptional activators enhanced rDNA transcription. The results indicate that activation by UCEs is not interchangeable between genes transcribed by RNA pol I and II, respectively, and suggest that different molecular mechanisms mediate the synergistic action of distant control sequences of different classes of genes.

Animals↗

PubMed1989

3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction.

We have studied the sequence requirements for 3'-end formation of rDNA transcripts in a cell-free system and show that the generation of correct ends of mouse pre-rRNA is brought about by a two-step process that involves a bona fide termination reaction, followed by a specific trimming of the primary transcript by 10 nucleotides. We show that termination of mouse ribosomal gene transcription by RNA polymerase I (pol I) takes place in front of an 18-bp DNA sequence element (the 'Sal box'), which was previously shown to function as termination signal. Termination of pol I transcription occurs at a fixed distance (11 bp) upstream of the Sal box, independent of the sequence of adjacent gene regions. The processing reaction, however, is strongly influenced by sequences flanking the termination signal at the 5' site. Substitution of a cluster of T residues by guanines within the region of 3'-end formation abolishes the 3'-terminal trimming of the primary transcript. Interestingly, this 3'-terminal processing event, which can be uncoupled from the termination reaction, requires both a correct 3' end and specific sequences in the 3'-terminal region of the primary transcript. Read-through transcripts generated in the extract system or by SP6 RNA polymerase are no substrate for the processing nuclease(s). Because the termination and processing activity can be separated chromatographically, the nucleolytic activity does not reside in TTF-I, the factor that binds to the Sal box and directs transcription termination.

Animals↗

PubMed1988

The mouse ribosomal gene terminator consists of three functionally separable sequence elements.

The structural requirements for 3' end formation of mouse pre-rRNA have been studied. Three sequence elements are shown to be required for accurate and efficient transcription termination by RNA polymerase I (pol I) assayed both in a cell-free transcription system and in vivo after transfection of rDNA minigene constructs into 3T6 cells. The essential termination signal is the previously identified 18-bp conserved element (AGGTCGACCAGATTANTCCG) that contains a SalI restriction site. This sequence motif (the 'Sal box') interacts with a specific nuclear protein that directs transcription termination. Here we demonstrate that the 'Sal box' sequence motif is sufficient for termination of pol I transcripts and the release of the nascent RNA chains from the template. However, in addition to this termination signal, pyrimidine-rich sequences flanking the box at the 5' and 3' side play a role in the efficient and correct formation of authentic pre-rRNA termini. Downstream sequences contribute to the efficiency of the termination reaction, whereas the position of 3' end formation (i.e. 21 bp upstream of the 'Sal box') is affected by 5' flanking regions. These flanking regions are recognized by at least two different nuclear factors which specifically bind to DNA sequences located upstream and downstream of the 'Sal box'.

Animals↗

PubMed1988

Purification and characterization of TTFI, a factor that mediates termination of mouse ribosomal DNA transcription.

Termination of rRNA gene transcription is dependent on an 18-base-pair sequence motif, AGGTCGAC CAG AT TA NTCCG (the Sal box), which is present several times in the spacer region downstream of the 3' end of the pre-rRNA coding region. We report here the purification to molecular homogeneity of a nuclear factor which specifically interacts with the Sal box element. Addition of the isolated protein to S-100 extracts which contain low levels of the Sal box-binding protein and are therefore termination incompetent restores terminating activity, indicating that this protein is a polymerase I-specific transcription termination factor. The purified protein (termed TTFI) has a molecular weight of approximately 105,000 on sodium dodecyl sulfate-polyacrylamide gels. Mild proteolysis generates a relatively protease-resistant core which still specifically recognizes its target sequence. However, the termination activity has been lost, suggesting that the interaction with the DNA and the interaction with the transcription apparatus reside in different protein domains.

Animals↗

PubMed1987

Natural point mutations within rat rDNA transcription terminator elements reveal the functional importance of single bases for factor binding and termination.

The rat rDNA transcription unit extends 560-565 bp into the spacer downstream of the 28S rRNA coding region. The site of 3' end formation is located in front of a conserved 18 bp sequence element which is repeated eight times in the 3' spacer between nucleotides +582 and +1767 relative to the 3' terminus of 28S rRNA. These sequence motifs are almost identical to the RNA polymerase I transcription termination signal (the Sal I box) that has previously been identified in the 3' terminal spacer of mouse rDNA. Interestingly, each of the single rat elements contains one or more base substitutions as compared to the murine Sal I box. Individual rat Sal I boxes were cloned and tested for their ability to interact with the murine termination factor and to direct transcription termination. It is shown that five of the eight boxes represent genuine transcription terminators, while three elements contain certain point mutations which are not recognized by the nuclear Sal I box-binding protein and therefore are functionally inactive.Images

Animals↗

PubMed1987

A novel promoter in the mouse rDNA spacer is active in vivo and in vitro.

We have identified a novel RNA polymerase I (pol I) transcription initiation site within the 'non-transcribed' spacer of mouse rDNA. This spacer promoter is located about 2 kb upstream of the 45S pre-rRNA promoter and directs specific transcription initiations both in a cell-free system using truncated templates and in vivo after transfection into mouse cells. The spacer promoter contains an 11 out of 16 bases match to the core element of the major ribosomal gene promoter and is oriented in the same direction. It exerts a significantly lower transcriptional activity as compared to the 45S pre-rRNA promoter. The elongation of transcripts initiated at the spacer promoter is stopped at a termination signal located 170 bp upstream of the pre-rRNA start site. Since it has been previously shown that, in addition to its terminator function, the same sequence motif acts as an upstream element of the adjacent gene promoter, the function of the spacer promoter may be to capture free pol I molecules and drive them to the gene promoter in order to achieve the high level of transcription characteristic of eukaryotic rRNA genes.

Animals↗

PubMed1987

Evolutionary changes of sequences and factors that direct transcription termination of human and mouse ribsomal genes.

We have analyzed the sequences required for termination of human rDNA transcription. The human ribosomal transcription unit is shown to extend about 350 nucleotides into the 3'-terminal spacer and ends immediately upstream of a region with a distinct sequence heterogeneity. This heterogeneous region contains a cluster of conserved 10-base pair sequence elements which exert a striking homology to the proximal part of the 18-base pair murine rDNA transcription termination signal sequence, termed SalI box. Exonuclease III protection assays and in vitro transcription experiments with both homologous and heterologous human-mouse minigene constructs, and extracts from HeLa or Ehrlich ascites cells, reveal a functional analogy of the human sequence to the mouse SalI box. It mediates binding of a nuclear protein which functions as a transcription termination factor. The murine signal sequence is recognized by the human factor but not vice versa. The different sequence specificities and electrophoretic properties of the functionally equivalent protein factors suggest that a molecular coevolution has taken place between the termination signal sequences and the genes coding for the termination factors.

Animals↗

PubMed1986

A transcription terminator located upstream of the mouse rDNA initiation site affects rRNA synthesis.

Mouse ribosomal genes have a short sequence upstream of the transcription initiation site that is related in structure and function to the terminator boxes previously identified at the 3' end of the transcription unit. This upstream terminator recognizes the same protein factor as the 3'-terminal sites and is able to terminate RNA polymerase I transcription in vitro. S1 mapping and nucleolar run-on experiments reveal the presence of 5'-terminal spacer transcripts that are terminated at this site. These transcripts probably derive from spacer promoters, one of which has been identified approximately 2 kb upstream of the pre-rRNA start site. The interaction of a specific nuclear factor with the upstream terminator increases the efficiency of initiation, suggesting that transcription termination and initiation at the adjacent promoter work in an interrelated fashion.

Animals↗