T-Rex: Assignment 2: Article Summaries

X-Ray Structure of a Rex-Family Repressor

The Rex repressor derived from Thermus aquaticus (T-Rex) regulates respiratory genes based on the NADH/NAD+ flux of the cell. In this paper, a 2.9A resolution X-ray structure of T-rex bound to NADH helps determine structural features of this mechanism. Both reduced and oxidized forms of NAD(H) binds to T-Rex, however NADH inhibits DNA binding while NAD+ allows it. The Rex operator (ROP) is the target site of DNA and is found upstream of many major respiratory protein encoding genes. Low levels of oxygen are through to cause an increase in NADH, which replaces the NAD+ bound to T-Rex and inhibits DNA from binding. With DNA released from the repressor, expression of genes crucial to respiration function will be encoded. Figure below shows the DNA bound state of T-Rex to the ROP, with NAD+ bound; replacing NAD+ with NADH will remove T-Rex from operator.

Structural Basis for NADH/NAD+ Redox Sensing by a Rex-Family Repressor

Rex-family repressors are mostly found in gram-positive bacteria, where they fine-tune the expression of genes through both the binding of NADH and NAD+. This makes the Rex repressors unique as they have more control over regulation as opposed to if they could just sense NADH levels alone. Additionally, T-Rex is unique in that it its cofactor NAD" is not only able to inhibit DNA binding, but also competes with NADH to do so. In this paper, structures were determined for T-Rex while bound to NAD+, the DNA operator, and without a ligand. Crystallization and Suface Plasmon Resonance studies helped reveal a rotation in the subunits upon NADH binding, which prevents continuation of DNA binding, a process at the heart of this regulation.

Oxygen, Metabolism, and Gene Expression: The T-Rex Connection

The Rex-family receptors have helped determine how gram-positive bacterial species modulate their gene expression response under limited oxygen conditions. NADH-bound T-rex has two domains: a dinucleotide binding domain and a winged-helix DNA binding domain (see figure below). There are three ways T-rex's structure helps decipher the mechanism by which NADH/NAD+ exchange changes the affinity of DNA to dimeric T-rex. First, when NADH binds to the winged helix, T-Rex dissociates from DNA because the binding domain arrangement does not allow T-Rex to bind to its Rex operator. Secondly, exchange of NADH for NAD+ brings a favorable conformation change in the wing-helices. And finally, upon the NADH/NAD+ exchange, the C-terminus of the alpha-helix in the monomer serves as a level that inserts between the dinucleotide and winged helices domain. These structural considerations give an idea of how NADH/NAD+ regulates gene expression.