Structural basis for specific DNA sequence motif recognition by the TFAP2 transcription factors.

The TFAP2 family regulates gene expression during differentiation, development, and organogenesis, and includes five homologs in humans. They all possess a highly conserved DNA binding domain (DBD) followed by a helix-span-helix (HSH) domain. The DBD-HSH tandem domain specifically binds to a GCC(N3)GGC consensus sequence, but the precise recognition mechanisms remain unclear. Here, we found that TFAP2 preferred binding to the GCC(N3)GGC sequence, and the pseudo-palindromic GCC and GGC motifs and the length of the central spacer between the two motifs determined their binding specificity. Structural studies revealed that the two flat amphipathic α-helical HSH domains of TFAP2A stacked with each other to form a dimer via hydrophobic interactions, while the stabilized loops from both DBD domains inserted into two neighboring major grooves of the DNA duplex to form base-specific interactions. This specific DNA binding mechanism controlled the length of the central spacer and determined the DNA sequence specificity of TFAP2. Mutations of the TFAP2 proteins are implicated in various diseases. We illustrated that reduction or disruption of the DNA binding ability of the TFAP2 proteins is the primary cause of TFAP2 mutation-associated diseases. Thus, our findings also offer valuable insights into the pathogenesis of disease-associated mutations in TFAP2 proteins.

Effects of Four Strains of Actinomycetes on the Content of Terpenoids in Baijiu.

Terpenoids not only are an important health factor in baijiu but also contribute to the elegance and finesse of baijiu, and actinomycetes act as an important source of terpenoids in baijiu. Four strains of actinomycetes-Streptomyces violascens (SPQ1), S. sampsonii (SPS1), S. thermophilus (SPG1), and S. griseus (SPH1)-obtained from the Daqu, pit mud, fermented grains and air, respectively, in the production of baijiu were used in solid-state and liquid fermentation with five brewing raw materials as the substrates. The terpenoids in the metabolites were analyzed and compared using gas chromatography-mass spectrometry (GC-MS). We found that the four strains of actinomycetes produced 31 terpenoids from the hydrolysates of five fermentation substrates during liquid fermentation, and the total terpenoid content was 989.94 µg/kg in the fermentation products. After 28 days of solid-state fermentation, the four actinomycete strains produced 64 terpenoids using the five fermentation substrates, and the total terpenoid content was 23,651.52 µg/kg in the fermentation products. The different fermentation substrates and fermentation methods have a great influence on the terpenoids produced by actinomycetes.

Structural basis for the recognition of the S2, S5-phosphorylated RNA polymerase II CTD by the mRNA anti-terminator protein hSCAF4.

The C-terminal domain (CTD) of RNA polymerase II serves as a binding platform for numerous enzymes and transcription factors involved in nascent RNA processing and the transcription cycle. The S2, S5-phosphorylated CTD is recognized by the transcription factor SCAF4, which functions as a transcription anti-terminator by preventing early mRNA transcript cleavage and polyadenylation. Here, we measured the binding affinities of differently modified CTD peptides by hSCAF4 and solved the complex structure of the hSCAF4-CTD-interaction domain (CID) bound to a S2, S5-quadra-phosphorylated CTD peptide. Our results revealed that the S2, S5-quadra-phosphorylated CTD peptide adopts a trans conformation and is located in a positively charged binding groove of hSCAF4-CID. Although hSCAF4-CID has almost the same binding pattern to the CTD as other CID-containing proteins, it preferentially binds to the S2, S5-phosphorylated CTD. Our findings provide insight into the regulatory mechanism of hSCAF4 in transcription termination.