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[Cys.sub.2][His.sub.2] zinc finger proteins make up the largest class of transcription factors encoded in the genomes of higher eukaryotes. Recent studies of the Ikaros transcription factor demonstrated that this zinc finger protein undergoes cell cycle-dependent changes in association with DNA that seem to be due to phosphorylation of Thr or Ser residues in the linker regions connecting adjacent zinc finger domains. The high degree of conservation of this linker sequence within the [Cys.sub.2][His.sub.2] superfamily suggested a common mechanism for the cell cycle-dependent modulation of DNA-binding affinity throughout this large class of transcription factors. The effects of linker phosphorylation on DNA-binding affinity were investigated through a direct comparison of the DNA-binding properties of four synthetic zinc finger proteins produced by native chemical ligation. The four proteins, comprising three zinc finger domains joined by two consensus Thr-Gly-Glu-Lys-Pro linkers, correspond to all four possible combinations of linker Thr phosphorylation states. Fluorescence-based DNA-binding studies of specific DNA-binding site revealed that phosphorylation of a single linker reduced binding affinity [approximately equal to] 40-fold, whereas phosphorylation of both linkers reduced binding affinity 130-fold. These result with purified components demonstrate that linker phosphorylation does, indeed, produce a significant reduction in DNA-binding affinity and support a model wherein a single cell cycle-dependent Ser/Thr kinase could simultaneously inactivate a large number of zinc finger transcription factors.

Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. Nup475 contains two tandemly repeated sequences YKTELC[X.sub.8]C[X.sub.5]C[X.sub.3]H ([Cys.sub.3]His repeats) that are thought to be zinc-binding domains. Similar sequences have been found in a number of proteins from various species of eukaryotes. To determine the metal binding properties and secondary structure of the putative zinc-binding domains of Nup475, we have used synthetic or recombinant peptides that contain one or two domain sequences. The peptide with a single domain bound 1.0 [+ or -] 0.1 equivalents of [Co.sup.2+], and the peptide with two domains bound 1.7 [+ or -] 0.4 equivalents of [Co.sup.2+]. Both peptides bound [Co.sup.2+] and [Zn.sup.z+] with affinities similar to those of classical zinc finger peptides. In each case, the [Co.sup.2+] complex exhibited strong d-d transitions characteristic of tetrahedral coordination. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second [Cys.sub.3]His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first [Cys.sub.3]His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.

A new oligonucleotide library search procedure represents the sequence of each member by the length of the associated DNA portion and enables binding-site detection through denaturing gel electrophoresis. As this method allows the parallel evaluation of all the member ligands, estimates of the relative suitability of all the ligands can be obtained. The method is used for a series of 18 lys2His2 zinc finger proteins flanked by two constant domains.

A protocol to design zinc-finger proteins with different DNA binding specificities is presented. The designed protein consists of three individually modified zinc fingers differing in one to four residues of the recognition region, which spans seven residues. Characterization of different designed proteins showed that the zinc-finger domains exhibit qualitatively modular behavior.

Zinc finger domain sequences were analyzed to convert the deoxyribonucleic acid binding domain of sp1 using three amino acid changes in the second zinc finger domain. It was shown that it is possible to make additional changes in DNA binding specificity by a systematic variation of three contact positions. Results of this study provide a code that will facilitate the construction of zinc finger proteins with preselected DNA site specificity.

A polyanine 26- amino acid peptide characteristic of the TFIIIA-type zinc finger domains has been synthesized and characterized using its metal binding and structural properties. Results show that the peptide folds in the presence of appropriate metal ions to form a complex that has a three-dimensional structure similar to zinc finger domains with more natural sequences. The sequences form a 2:1 peptide/metal complex in which the cysteinate ligands are coordinated to the metal ion but not to histidine.

The DNA-binding properties of fragments of the zinc finger protein Sp1 and other zinc finger proteins with guanine-richbinding sites were described. Studies have shown that Sp1 peptide fragment withthe binding site 5'-GGGGCGGGGC-3' can bind specific regions of DNA in a manner similar to intact Sp1. Further studies should determine whether rules can be developed that would allow deduction of the preferred DNA binding sites of zincfinger domains based on zinc finger amino acid sequences.

The US research enterprise is under significant strain due to stagnant funding, an expanding workforce, and complex regulations that increase costs and slow the pace of research. In response, a number of groups have analyzed the problems and offered recommendations for resolving these issues. However, many of these recommendations lacked follow-up implementation, allowing the damage of stagnant funding and outdated policies to persist. Here, we analyze nine reports published since the beginning of 2012 and consolidate over 250 suggestions into eight consensus recommendations made by the majority of the reports. We then propose how to implement these consensus recommendations, and we identify critical issues, such as improving workforce diversity and stakeholder interactions, on which the community has yet to achieve consensus. [ABSTRACT FROM AUTHOR]