Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg–SHP and Rgg–target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Streptococcus dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg–DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure–function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.

葡萄糖基转移酶（Rgg）家族蛋白的调节基因，如Rgg2和Rgg3，已作为链球菌中主要的群体感应调节转录因子出现，可控制毒力，抗微生物性和生物膜形成。Rgg2和Rgg3的功能受到它们与称为短疏水肽（SHP）的寡肽群体感应信号相互作用的调节。Rgg–SHP和Rgg–靶标DNA启动子特异性的分子基础尚不清楚。为了缩小这一差距，我们确定了嗜热链球菌的低温电子显微镜（cryo-EM）结构Rgg3绑定到其群体感应信号SHP3和Rgg3的X射线晶体结构。这些结构与Rgg与环孢菌素A（CsA）（一种SHP诱导的Rgg活性抑制剂）的复合物的比较，揭示了CsA功能的分子基础。此外，为了确定Rgg蛋白如何识别DNA启动子，我们确定了dysgalactiae链球菌Rgg2和嗜热链球菌的X射线晶体结构。Rgg3与它们的靶DNA启动子复合。观察到的Rgg-DNA相互作用的生理重要性已使用体内遗传实验和体外生化分析进行了剖析。基于这些结构功能研究，我们提出了Rgg调节相互作用的修订统一模型。与现有模型相反，在现有模型中Rgg2蛋白是转录激活因子，而Rgg3蛋白是转录抑制因子，我们建议两者均具有转录激活能力。但是，当具有不同激活要求的Rgg蛋白竞争相同的DNA启动子时，具有更严格激活要求的Rgg蛋白通过阻止SHP结合的构象活性Rgg蛋白的启动子进入而起阻遏物的作用。尽管以前没有描述过类似的基因表达调控方案，