RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , RNA Polimerase I/metabolismo , Transcrição Gênica/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Nucléolo Celular/química , Clonagem Molecular , DNA/metabolismo , DNA Ribossômico/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência , Análise de Sequência de DNA , Deleção de Sequência/genética , Homologia de Sequência de Aminoácidos , Regiões Terminadoras Genéticas/genética , Fatores de Transcrição , Triptofano/metabolismoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , RNA Polimerase I/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Proteínas de Xenopus , Western Blotting , Candida/genética , Linhagem Celular , Humanos , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-BoxRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição , Processamento de Proteína Pós-Traducional , RNA Polimerase I/metabolismo , TATA Box , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Carcinoma de Ehrlich/metabolismo , Cromatografia de Afinidade , Sequência Conservada , Proteínas de Ligação a DNA/isolamento & purificação , Expressão Gênica , Células HeLa , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Deleção de Sequência , Proteína de Ligação a TATA-Box , Fatores de Transcrição/isolamento & purificação , Transcrição GênicaRESUMO
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.
