Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction. 271 50

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.
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PMID:The amino-terminal domain of the transcription termination factor TTF-I causes protein oligomerization and inhibition of DNA binding. 887 44

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.
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PMID:Identification of a transcript release activity acting on ternary transcription complexes containing murine RNA polymerase I. 900 77

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.
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PMID:RNA polymerase I transcription on nucleosomal templates: the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression. 904 5

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.
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PMID:Oligomerization of the transcription termination factor TTF-I: implications for the structural organization of ribosomal transcription units. 909 22

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.
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PMID:RNA polymerase I transcription termination: similar mechanisms are employed by yeast and mammals. 915 65

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.
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PMID:Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I. 926 35

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.
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PMID:Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes. 958 79

Transcription termination by RNA polymerase I (Pol I) is a stepwise process. First the elongating RNA polymerase is forced to pause by DNA-bound transcription termination factor (TTF-I). Then the ternary transcription complex is dissociated by PTRF, a novel factor that promotes release of both nascent transcripts and Pol I from the template. In this study we have investigated the ability of PTRF to liberate transcripts from ternary transcription complexes isolated from yeast and mouse. Using immobilized, tailed templates that contain terminator sequences from Saccharomyces cerevisiae and mouse, respectively, we demonstrate that PTRF promotes release of terminated transcripts, irrespective of whether mouse Pol I has interacted with the murine termination factor TTF-I or its yeast homolog Reb1p. In contrast, mouse Pol I paused by the lac repressor remains bound to the template both in the presence and absence of PTRF. We demonstrate that PTRF interacts with the largest subunit of murine Pol I, with TTF-I and Reb1p, but not the lac repressor. The results imply that Pol I transcription termination in yeast and mouse is mediated by conserved interactions between Pol I, Reb1p/TTF-I and PTRF.
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PMID:Mechanism of transcription termination: PTRF interacts with the largest subunit of RNA polymerase I and dissociates paused transcription complexes from yeast and mouse. 1058 39

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.
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PMID:The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I. 1113 12


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