Data Sheet 1_WhiA transcription factor provides feedback loop between translation and energy production in a genome-reduced bacterium.pdf

IntroductionWhiA is a conserved protein found in numerous bacteria. It consists of an HTH DNA-binding domain linked with a homing endonuclease (HEN) domain. WhiA is one of the most conserved transcription factors in reduced bacteria of the class Mollicutes. Its function in Mollicutes is unknown, while it is well-characterized in Streptomyces. Here, we focused on WhiA protein from Mycoplasma gallisepticum.
MethodsWe used a combination molecular dynamics, EMSA, MST and AFM to study the DNA-binding and ATP-binding properties of WhiA from M. gallisepticum. The transcriptional repressor function of WhiA was demonstrated using gene knockdown, reporter constructs and proteome analysis.
ResultsWe demonstrate that WhiA homolog from M. gallisepticum binds a conserved sequence of the GAYACRCY core (Y = C or T, R = A or G), which is located in the promoter of an operon coding for ribosomal proteins and adenylate kinase (rpsJ operon). We show that WhiA in M. gallisepticum is a repressor of rpsJ operon and a sensor of ATP. HTH domain binds to the core motif and HEN domain binds to the auxiliary motif GTTGT that is located downstream to the core motif. We show that binding by both domains to DNA is required to fulfill the transcription repressor function. Knockdown of whiA does not affect actively growing M. gallisepticum, but leads to the growth retardation after freezing.
DiscussionWe propose the following model for M. gallisepticum WhiA function. WhiA remains bound to the core motif at any conditions. At low ATP concentrations (starvation) HEN domain binds auxiliary motif and represses rpsJ operon transcription. At high ATP concentrations (nutrient-rich conditions) HEN domain binds ATP and releases auxiliary motif. It leads to the de-repression of rpsJ operon and increased production of ribosomal proteins.

引言
WhiA是一类广泛分布于多种细菌中的保守蛋白，由螺旋-转角-螺旋（helix-turn-helix, HTH）DNA结合结构域与归巢核酸内切酶（homing endonuclease, HEN）结构域融合而成。WhiA是柔膜菌纲（Mollicutes）简化型细菌中最为保守的转录因子之一。目前其在柔膜菌纲细菌中的功能尚不明确，但在链霉菌属（Streptomyces）中其功能已得到充分解析。本研究聚焦于鸡毒支原体（Mycoplasma gallisepticum）来源的WhiA蛋白。
方法
我们联合运用分子动力学、电泳迁移率变动分析（Electrophoretic Mobility Shift Assay, EMSA）、微量热泳动（MicroScale Thermophoresis, MST）以及原子力显微镜（Atomic Force Microscopy, AFM）技术，对鸡毒支原体WhiA的DNA结合与ATP结合特性进行研究。通过基因敲低、报告基因构建体与蛋白质组分析，验证了WhiA的转录阻遏功能。
结果
本研究证实，鸡毒支原体的WhiA同源蛋白可结合一段保守的GAYACRCY核心基序（其中Y代表C或T，R代表A或G），该基序位于编码核糖体蛋白与腺苷酸激酶的操纵子（rpsJ操纵子）的启动子区域。实验表明，鸡毒支原体中的WhiA既是rpsJ操纵子的转录阻遏蛋白，同时也是ATP的传感器。HTH结构域可结合核心基序，而HEN结构域则结合位于核心基序下游的辅助基序GTTGT。研究证实，两个结构域与DNA的结合均为实现转录阻遏功能所必需。敲低whiA基因不会对活跃生长期的鸡毒支原体产生影响，但会导致其冻存后出现生长迟缓。
讨论
我们针对鸡毒支原体WhiA的功能提出如下模型：在所有条件下，WhiA均会结合核心基序。当ATP浓度较低（营养饥饿状态）时，HEN结构域会结合辅助基序，从而阻遏rpsJ操纵子的转录；当ATP浓度较高（营养富足条件）时，HEN结构域会结合ATP并释放辅助基序，进而解除对rpsJ操纵子的阻遏，提升核糖体蛋白的合成量。