RESUMO
The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis. The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at -87 to -84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended -10 promoter, it lacks a reasonable -35 element. Genetic analysis and structural simulations predicted that the absence of the -35 element might be compensated by interactions between σA and αCTD and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified, and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors, including the ubiquitous response regulators of two-component regulatory systems, particularly in Gram-positive bacteria.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Nitrito Redutases/genética , Fatores de Transcrição/genética , Ativação Transcricional , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrito Redutases/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
Although the requirement of pituitary-derived LH for ovulation is well documented, the intrafollicular paracrine and autocrine processes elicited by LH necessary for follicle rupture are not fully understood. Evaluating a published rhesus macaque periovulatory transcriptome database revealed that mRNA encoding leukemia inhibitory factor (LIF) and its downstream signaling effectors are up-regulated in the follicle after animals receive an ovulatory stimulus (human chorionic gonadotropin [hCG]). Follicular LIF mRNA and protein levels are below the limit of detection before the administration of hCG but increase significantly 12 hours thereafter. Downstream LIF receptor (LIFR) signaling components including IL-6 signal transducer, the receptor associated Janus kinase 1, and the transcription factor signal transducer and activator of transcription 3 also exhibit increased expression in the rhesus macaque follicle 12 hours after administration of an ovulatory hCG bolus. A laparoscopic ovarian evaluation 72 hours after the injection of a LIF antagonist (soluble LIFR) into the rhesus macaque preovulatory follicle and hCG administration revealed blocking LIF action prevented ovulation (typically occurs 36-44 h after hCG). Moreover, ovaries removed 52 hours after both hCG and intrafollicular soluble LIFR administration confirmed ovulation was blocked as evidenced by the presence of an intact follicle and a trapped cumulus-oocyte complex. These findings give new insight into the role of LIF in the primate ovary and could lead to the development of new approaches for the control of fertility.