https://www.cell.com/cell/pdf/S0092-8674(14)01036-8.pdf

Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity

▪ Abstract Cys2His2 zinc fingers are one of the most common DNA-binding motifs found in eukaryotic transcription factors. These proteins typically contain several fingers that make tandem contacts along the DNA. Each finger has a conserved ββα structure, and amino acids on the surface of the α-helix contact bases in the major groove. This simple, modular structure of zinc finger proteins, and the wide variety of DNA sequences they can recognize, make them an attractive framework for attempts to design novel DNA-binding proteins. Several studies have selected fingers with new specificities, and there clearly are recurring patterns in the observed side chain–base interactions. However, the structural details of recognition are intricate enough that there are no general rules (a “recognition code”) that would allow the design of an optimal protein for any desired target site. Construction of multifinger proteins is also complicated by interactions between neighboring fingers and the effect of the intervening...

https://www.nsm.buffalo.edu/~koudelka/piero/ZInc_fingers_by_Pabo.pdf.gz

DNA recognition by Cys2His2 zinc finger proteins.

The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins.

The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5.

DNA RECOGNITION BY Cys 2 His 2 ZINC FINGER PROTEINS

Nuclear receptors for steroid hormones, thyroid hormone, retinoids, and vitamin D are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription complex. In an effort to identify potential coregulators, receptor fusions with glutathione-S-transferase were used to isolate proteins in nuclear extracts capable of binding nuclear hormone receptors. Glutathione-S-transferase fusions with mouse retinoid X receptor-α enabled the selective isolation of a 65-kDa protein (p65) from nuclear extracts of rat and human cells. Binding of p65 to mouse retinoid X receptor-α was centered around the DNA-binding domain. p65 also bound regions encompassing the DNA-binding domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65 was identified as TLS (translocated-in-liposarcoma), a recently identified member of the RNP family of nuclear RNA-binding proteins whose members are thought to function in RNA proces...

A Rovelli

Papers

Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation.