A hydrophobic residue stabilizes dimers of regulatory ACT-like domains in plant basic helix-loop-helix transcription factors.

About a third of the plant basic helix-loop-helix (bHLH) transcription factors harbor a C-terminal aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain, which was originally identified in the maize R regulator of anthocyanin biosynthesis, where it modulates the ability of the bHLH to dimerize and bind DNA. Characterization of other bHLH ACT-like domains, such as the one in the Arabidopsis R ortholog, GL3, has not definitively confirmed dimerization, raising the question of the overall role of this potential regulatory domain. To learn more, we compared the dimerization of the ACT-like domains of R (RACT) and GL3 (GL3ACT). We show that RACT dimerizes with a dissociation constant around 100 nM, over an order of magnitude stronger than GL3ACT. Structural predictions combined with mutational analyses demonstrated that V568, located in a hydrophobic pocket in RACT, is important: when mutated to the Ser residue present in GL3ACT, dimerization affinity dropped by almost an order of magnitude. The converse S595V mutation in GL3ACT significantly increased the dimerization strength. We cloned and assayed dimerization for all identified maize ACT-like domains and determined that 12 of 42 formed heterodimers in yeast two-hybrid assays, irrespective of whether they harbored V568, which was often replaced by other aliphatic amino acids. Moreover, we determined that the presence of polar residues at that position occurs only in a small subset of anthocyanin regulators. The combined results provide new insights into possibly regulatory mechanisms and suggest that many of the other plant ACT-like domains associate to modulate fundamental cellular processes.

The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells.

We report that the cationic porphyrin TmPyP4, which is known mainly as a DNA G-quadruplex stabilizer, unfolds an unusually stable all purine RNA G-quadruplex (M3Q) that is located in the 5'-UTR of MT3-MMP mRNA. When the interaction between TmPyP4 and M3Q was monitored by UV spectroscopy a 22-nm bathochromic shift and 75% hypochromicity of the porphin major Soret band was observed indicating direct binding of the two molecules. TmPyP4 disrupts folded M3Q in a concentration-dependent fashion as was observed by circular dichroism (CD), 1D (1)H NMR and native gel electrophoresis. Additionally, when TmPyP4 is present during the folding process it inhibits the M3Q RNA from adopting a G-quadruplex structure. Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex. However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct. Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.