Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Purification of a model substrate for transcription factor phosphorylation by ERK2.

ERK2 belongs to the mitogen-activated protein kinase subfamily, which plays a pivotal role in cell signal transduction, in which it mediates effects on proliferation and differentiation by growth factors and hormones. While its cellular function has been under intense scrutiny since its initial discovery nearly 15 years ago, little progress has been made in understanding its kinetic mechanism. Such an understanding is important for the development of potent and specific inhibitors. A contributory factor has been the lack of a protein substrate suitable for rigorous mechanistic studies. Here we report the expression, purification, and characterization of the N-terminus (residues 1 through 138) of the transcription factor Ets-1, an excellent model substrate for ERK2 mechanistic studies. (His(6)-tagged)Ets-1(1-138) was expressed in Escherichia coli and rapidly purified in two steps by nickel-agarose-affinity chromatography, followed by high-resolution Mono-Q anion-exchange chromatography. A yield of 60 mg of the purified protein per liter of culture was obtained and could be stored conveniently at -80 degrees C in water. Rigorous characterization demonstrated that under the assay conditions, (His(6)-tagged)Ets-1(1-138) is exclusively phosphorylated on residue Thr-38 by ERK2 with the following Michaelis parameters: k(cat) = 17 s(-1), K(ATP)(m) = 140 microM, K(ATP)(i) = 68 microM, K(Ets-1)(m) = 19 microM, and K(Ets-1)(i) = 9.3 microM.

The kinetic mechanism of the dual phosphorylation of the ATF2 transcription factor by p38 mitogen-activated protein (MAP) kinase alpha. Implications for signal/response profiles of MAP kinase pathways.

The mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 of the glutathione S-transferase fusion protein of the trans-activation domain of activating transcription factor-2 (GST-ATF2-(1-115)) by p38 MAPKalpha (p38alpha) as a model system for the phosphorylation of ATF2 by p38alpha. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38alpha dissociates from GST-ATF2-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38alpha showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced the specificity, k(cat)/K(M), of p38alpha for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38alpha in vivo is essentially Michaelian and provides insight into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate.