ABSTRACT
The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions -9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions -5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.
Subject(s)
Promoter Regions, Genetic , Transcription Factors, TFII , Transcription Factors/genetics , Transcription, Genetic , Xeroderma Pigmentosum/genetics , Adenosine Triphosphate/metabolism , Base Sequence , Blotting, Western , Cross-Linking Reagents , DNA/metabolism , Gene Deletion , HeLa Cells , Humans , Hydrolysis , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Proteins/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Nucleic Acid , Transcription Factor TFIIHABSTRACT
DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK/ZPK in mammalian cells. Because analysis of subcellular distribution may provide important clues concerning the potential in vivo function of a protein, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. Light microscopic immunocytochemistry experiments performed with the antiserum revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear structure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatus, caused disruption of DLK/MUK/ZPK perinuclear staining. Ultrastructural observation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses revealed that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aqueous phase when membranes were subjected to Triton X-114 extraction. On the basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral membrane protein tightly associated with the cytoplasmic face of the Golgi apparatus. (J Histochem Cytochem 47:1287-1296, 1999)
Subject(s)
Golgi Apparatus/metabolism , MAP Kinase Kinase Kinases , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , 3T3 Cells/drug effects , 3T3 Cells/metabolism , Animals , Antibody Specificity , Brefeldin A/pharmacology , Cell Membrane/metabolism , Golgi Apparatus/chemistry , Golgi Apparatus/drug effects , Immunoblotting , Immunohistochemistry , Mice , Microscopy, Immunoelectron , Protein Kinases/isolation & purification , Protein Serine-Threonine Kinases/isolation & purification , Subcellular Fractions/chemistry , Subcellular Fractions/metabolismABSTRACT
The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) functions at multiple stages of transcription and is involved in the coupling of transcription to pre-mRNA processing. We have used site-specific protein-DNA photocross-linking to determine the position of the CTD along promoter DNA in the transcriptional pre-initiation complex. Comparison of the promoter contacts made by RNAP II with or without the CTD indicate that the CTD approaches promoter DNA downstream of the transcriptional initiation site between positions +16 and +26. Incubation of pre-assembled initiation complexes with antibodies to the CTD prior to UV irradiation led to specific photocross-linking of the IgG heavy chain to nucleotide +17, indicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjunction with previously published observations, our structural data are fully compatible with the notion that DNA wrapping around RNAP II places the CTD and the RNA exit channel into juxtaposition and provide a rationale for contacts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.
Subject(s)
Nucleic Acid Conformation , RNA Polymerase II/chemistry , Affinity Labels , Animals , Base Sequence , Cattle , Cross-Linking Reagents , DNA/chemistry , Immunoglobulin Heavy Chains/chemistry , Models, Molecular , Molecular Sequence Data , Photolysis , Promoter Regions, Genetic , RNA/chemistry , RNA Precursors/genetics , Thymus Gland/enzymology , Transcription Factors/genetics , Transcription, Genetic , Ultraviolet RaysABSTRACT
The formation of the RNA polymerase II (Pol II) initiation complex was analyzed using site-specific protein-DNA photo-cross-linking. We show that the RAP74 subunit of transcription factor (TF) IIF, through its RAP30-binding domain and an adjacent region necessary for the formation of homomeric interactions in vitro, dramatically alters the distribution of RAP30, TFIIE, and Pol II along promoter DNA between positions -40 and +26. This isomerization of the complex, which requires both TFIIF and TFIIE, is accompanied by tight wrapping of the promoter DNA for almost a full turn around Pol II. Addition of TFIIH enhances photo-cross-linking of Pol II to a number of promoter positions, suggesting that TFIIH tightens the DNA wrap around the enzyme. We present a general model to describe transcription initiation.
Subject(s)
Promoter Regions, Genetic , RNA Polymerase II/metabolism , Transcription Factors, TFII , Transcription Factors/metabolism , Adenoviridae/genetics , Base Sequence , Binding Sites , Cross-Linking Reagents , DNA, Viral/chemistry , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , RNA Polymerase II/chemistry , Recombinant Proteins , Transcription Factors/chemistryABSTRACT
ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.
Subject(s)
Liver Regeneration/genetics , Liver Regeneration/physiology , MAP Kinase Kinase Kinases , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Gene Expression , Hepatectomy , In Situ Hybridization , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tissue DistributionABSTRACT
ZPK is a recently described serine/threonine protein kinase that is thought to be involved in the regulation of cell proliferation and differentiation. To directly determine whether ZPK exhibits any effect on cell growth, NIH 3T3 fibroblasts were transfected with an expression vector harboring the murine ZPK cDNA. Stable expression of this construct led to a dramatic reduction in the proliferative capacity of these cells as measured by a colony formation assay in monolayer culture. By contrast, overexpression of a ZPK cDNA with a mutation in the ATP-binding domain did not affect clonal expansion of the transfected cells. These findings suggest that the ZPK gene may act as a negative regulator of cell growth and that this function may be mediated in part by the intrinsic kinase activity of the ZPK protein.
