Various ZnF motifs will bind DNA, usually in a sequence-specific manner. DNA-binding ZnF proteins are involved in transcriptional processes. Many transcription factors make DNA contacts to gene promoters via ZnF domains, some of which are classical C2H2 ZnF motifs. ZnF motifs typically wrap around the outside of a DNA double helix, following a right-handed helical path. Multiple DNA contacts are made, often involving more than one ZnF domain. These contacts are usually made to bases in the major DNA groove; for example, C2H2 ZnFs recognise DNA sequences by binding to the major groove of DNA via a short alpha-helix in the ZnF, the Znf spanning 3-4 bases of the DNA.
There are many DNA-binding proteins with ZnF motifs, several of which are involved in gene transcription. For example, transcription factor IIIA (TFIIIA) is an essential component of the initiation complex that interacts with separate specific transcription signals located within the ribosomal 5S RNA gene promoter. Non-binding spacers within TFIIIA allow it to span long DNA sequences, binding only to specific regions within the DNA.
Several synthetic ZnF proteins have been constructed as novel DNA-binding proteins with specific DNA recognition properties.
Various ZnF motifs will bind to RNA, including classical C2H2, CCHC and CCCH ZnFs, which are often involved in transcriptional and translational processes. The mode of RNA recognition differs from that of DNA recognition, and involves many different kinds of amino acid-nucleotide interactions, including backbone recognition of the RNA major groove as well as RNA base and loop recognition. For example, binding to dsRNA involves contacts with phosphates in the RNA backbone, as well as hydrophobic stacking interactions with specific nucleotide bases.
Some RNA-binding ZnF-containing proteins are involved in mRNA trafficking, activity and stability in eukaryotes, often recognising and binding to AU-rich sequences in the 3’-untranslated region of mRNAs. For example, the early response gene product TIS11d binds to AU-rich elements of tumour necrosis factor alpha-mRNA, which destabilises the mRNA. The CCCH ZnF motif in TIS11d recognises the target RNA sequence through hydrophobic stacking of a Tyr side-chain with a UU dinucleotide, and a Phe side-chain with a UU dinucleotide, which disrupts normal nucleotide stacking to kink the RNA.
Proteins carrying RNA-binding ZnF motifs include:
· Several retroviral proteins such as HIV nucleocapsid and reovirus sigma3
· Plant HUA1 nuclear protein
· Parasite proteins such as trypanosome tcZFP1 and leishmania mitochondrial RET1 uridylyl transferase
· Mammalian nucleolus proteins Wig-1 and JAZ
· Histone pre-mRNA processing proteins hZFP100 and tristetraprolin
Some ZnF domains act as protein-binding modules. Some of these domains contain ‘classical’ ZnF motifs more associated with DNA binding functions, although the interface used for protein-binding may differ from that used for nucleic acid-binding. In addition, several LIM, MYND, RING and PHD ZnF domains function in protein-protein interactions. Protein-binding ZnF proteins are involved in protein-protein interactions, chromatin remodelling, protein chaperoning, and zinc sensing.
Many protein-protein interactions involve ZnF-containing transcription factors. In some cases, these interactions are required to mediate the self-association of transcription factors, which can be a necessary step towards a functional complex. For example, Ikaros transcription factors require ZnF-mediated self-assocaition for high-affinity DNA-binding. Ikaros transcription factors often contain ones cluster of ZnF domain involved in DNA-binding and a second cluster involved in protein-protein interactions.
Protein-protein interactions involving transcription factors can also affect the regulation of gene expression. Proteins involved in transcription regulation often contain multiple ZnF domains, and have both DNA and protein-binding capacities. Protein-protein binding can function in the regulation of these factors, which in turn impacts gene regulation. For example, the transcription factor GATA-1 and the transcription regulator FOG-1 (Friend of GATA-1) both contain ZnF domains, which are involved in DNA and protein recognition and binding. GATA-1 binds in a sequence-specific manner to gene promoters. The binding of FOG-1 via ZnF motifs to GATA-1 alters gene expression from those promoters, and is essential for normal erythropoiesis in mammals. In addition, FOG-1 can interact with TACC3 (Transforming Acidic Coiled-Coil protein 3), where TACC3 can inhibit the nuclear localisation of FOG-1, which in turn impacts gene expression. GATA-1 itself appears capable of binding many different partners, including CBP, Fli-1, Sp1, EKLF and PU.1, each modifying the function of GATA-1.
ZnF-mediated protein-protein interactions involve other functions as well, including cytoskeleton organisation and development, epithelial development and cell adhesion. For example, the LIM-containing adaptor protein PINCH is involved in cell adhesion, growth and differentiation, interacting with the integrin signalling proteins ILK (integrin-like kinase) and Nck2. Other ZnF domains bind ubiquitin, such as RanBP-type and A20-type ZnF motifs, or are involved in protein ubiquitination, such as RING domain-containing E3 ligases. PHD domain-containing proteins are often involved in regulating chromatin structure, such as ING2 and BPTF that cooperate with p53 to induce cellular growth arrest and apoptosis, while others are able to recognise histones.