ABSTRACT
Interferons (IFNs) are a family of multifunctional proteins involved in immune activation, regulation of cell growth and antiviral response. They exert their functions by induction of several IFN-stimulated genes, including IFN regulatory factors (IRFs), a family of transcriptional regulators. One of these factors, IRF-2, was initially cloned as an antagonistic counterpart to IRF-1 with oncogenic potential. Here we describe a second isoform of IRF-2, termed IRF-2s, cloned from human and murine cells. This isoform lacks two amino acids located C-terminal of the DNA-binding domain, which is conserved in all IRF family members, leading to a change in the predicted secondary structure. Both isoforms have similar binding affinities to known target sequences in electrophoretic mobility shift assays. Using reporter gene constructs with the type IV promoter region of the MHC class II transactivator (CIITA), which is the essential factor for IFN-gamma-induced MHC class II expression, we show that the short isoform IRF-2s exhibits a weaker activation ability compared to IRF-2. Thus, our data present the first evidence of two IRF-2 isoforms with different regulatory ability.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins , Repressor Proteins , Transcription Factors , Transcriptional Activation , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , DNA/genetics , DNA-Binding Proteins/genetics , Genes, MHC Class II/genetics , Genes, Reporter , Humans , Interferon Regulatory Factor-2 , Leydig Cells/metabolism , Male , Mice , Molecular Sequence Data , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Secondary , RNA, Messenger/analysis , RNA, Messenger/genetics , Sequence Alignment , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , TransfectionABSTRACT
Double-stranded RNA adenosine deaminase (ADAR1) is an ubiquitous enzyme in metazoa that edits pre-mRNA changing adenosine to inosine in regions of double-stranded RNA. Zalpha, an N-terminal domain of human ADAR1 encompassing 76 amino acid residues, shows apparent specificity for the left-handed Z-DNA conformation adopted by alternating (dGdC) polymers modified by bromination or methylation, as well as for (dGdC)13 inserts present in supercoiled plasmids. Here, a combination of circular dichroism, fluorescence, and gel-retardation studies is utilized to characterize recombinant Zalpha peptide and to examine its interaction with DNA. Results from laser-Raman spectroscopy experiments provide direct evidence for the existence of Z-DNA in peptide-DNA complexes.
Subject(s)
Adenosine Deaminase/chemistry , DNA/chemistry , RNA Editing , Adenosine Deaminase/metabolism , Amino Acid Sequence , Animals , Chickens , Circular Dichroism , DNA/metabolism , Deoxycytidine/chemistry , Deoxyguanosine/chemistry , Hot Temperature , Humans , Macromolecular Substances , Molecular Sequence Data , Nucleic Acid Conformation , Peptides/chemistry , Polydeoxyribonucleotides/chemistry , Protein Conformation , Protein Denaturation , RNA-Binding Proteins , Spectrometry, Fluorescence , Spectrum Analysis, RamanABSTRACT
Z-DNA, the left-handed conformer of DNA, is stabilized by the negative supercoiling generated during the movement of an RNA polymerase through a gene. Recently, we have shown that the editing enzyme ADAR1 (double-stranded RNA adenosine deaminase, type 1) has two Z-DNA binding motifs, Zalpha and Zbeta, the function of which is currently unknown. Here we show that a peptide containing the Zalpha motif binds with high affinity to Z-DNA as a dimer, that the binding site is no larger than 6 bp and that the Zalpha domain can flip a range of sequences, including d(TA)3, into the Z-DNAconformation. Evidence is also presented to show that Zalpha and Zbeta interact to form a functional DNA binding site. Studies with atomic force microscopy reveal that binding of Zalpha to supercoiled plasmids is associated with relaxation of the plasmid. Pronounced kinking of DNA is observed, and appears to be induced by binding of Zalpha. The results reported here support a model where the Z-DNA binding motifs target ADAR1 to regions of negative supercoiling in actively transcribing genes. In this situation, binding by Zalpha would be dependent upon the local level of negative superhelicity rather than the presence of any particular sequence.
Subject(s)
Adenosine Deaminase/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Binding Sites , Humans , Microscopy, Atomic Force , Protein Binding , Protein Conformation , RNA-Binding ProteinsABSTRACT
Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zalpha) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5' to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zalpha has allowed identification of motifs common to this class of nucleic acid binding domain.
