Ferguson ZNF Sandbox: Difference between revisions

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In TFIIIA, there are nine consecutive zinc fingers. Individual zinc fingers can be positioned in both the major groove and across the minor groove of DNA. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove. <scene name='Ferguson_ZNF_Sandbox/1tf6/2'>1tf6</scene> shows a dimer with 6 zinc fingers on each bound to a stretch of DNA. With this interaction, TFIIIA helps control the transcription of the gene for ribosomal RNA. The string of zinc fingers curls along the DNA or RNA strands, binding in the grooves and extending amino acids inwards to read the bases. A single zinc finger does not bind very tightly and can only recognize 2 or 3 base pairs, but several can be strung together, causing the group to bind more tightly and allows it to read longer DNA sequences. This modular approach is so appealing that researchers are currently trying to design artificial zinc fingers with different specificities<ref>PMID:2503871</ref>.

====DNA Binding in mutant zinc finger domains====

We have used this approach to study two mutants in the N-terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159. Two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild-type ADR1b and the two mutant zinc finger domains were determined using two-dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild-type ADR1b domain is presented in detail. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein-DNA interaction<ref>PMCID:PMC2142395</ref>.

Ferguson ZNF Sandbox: Difference between revisions

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 In TFIIIA, there are nine consecutive zinc fingers.  Individual zinc fingers can be positioned in both the major groove and across the minor groove of DNA. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.  <scene name='Ferguson_ZNF_Sandbox/1tf6/2'>1tf6</scene> shows a dimer with 6 zinc fingers on each bound to a stretch of DNA.  With this interaction, TFIIIA helps control the transcription of the gene for ribosomal RNA.  The string of zinc fingers curls along the DNA or RNA strands, binding in the grooves and extending amino acids inwards to read the bases. A single zinc finger does not bind very tightly and can only recognize 2 or 3 base pairs, but several can be strung together, causing the group to bind more tightly and allows it to read longer DNA sequences. This modular approach is so appealing that researchers are currently trying to design artificial zinc fingers with different specificities<ref>PMID:2503871</ref>.
+====DNA Binding in mutant zinc finger domains====
+We have used this approach to study two mutants in the N-terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159. Two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild-type ADR1b and the two mutant zinc finger domains were determined using two-dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild-type ADR1b domain is presented in detail. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein-DNA interaction<ref>PMCID:PMC2142395</ref>.