RESUMO
Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome (mitochondrial DNA) and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The in vitro re-constituted transcriptional complexes of Saccharomyces cerevisiae (yeast) and humans, aided with high-resolution structures and biochemical characterizations, have provided a deeper understanding of the mechanism and regulation of mitochondrial DNA transcription. In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.
Assuntos
DNA Mitocondrial/química , DNA Mitocondrial/genética , Regulação da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , DNA Mitocondrial/metabolismo , Humanos , Proteínas Mitocondriais/genética , Fatores de Transcrição/genéticaRESUMO
Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.
Assuntos
Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Trifosfato de Adenosina , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: VIA is a simple, inexpensive test widely advocated for resource-limited settings. Major limitation of VIA is its low specificity. HPV DNA testing can be used to triage VIA-positive women if the facilities are available. The major concern for such strategy would be whether sample collection after acetic acid wash will alter HPV test characteristics. This study aimed to evaluate whether samples for HPV testing by Hybrid Capture 2 (HC2) technology can be collected immediately after VIA without altering test performance. METHODS: Total 204 VIA-positive women were recruited. Cervical samples were collected for HC2 test before and after VIA at the same sitting by the same provider. The paired samples were analyzed at the same laboratory by the same technician in the same batch of testing. Agreement in HC2 results between pre-VIA and post-VIA samples was estimated using kappa statistics. All women had colposcopy and biopsies were obtained if colposcopy was suspicious of neoplasia. Sensitivity and specificity of HC2 test in detecting CIN2+ lesions were calculated using negative colposcopy or biopsy as the gold standard and were compared between the pre and post VIA samples. RESULTS: Almost perfect agreement in HC2 results (kappa=0.85) and RLU/Cut off ratios (correlation coefficient=0.92) was observed between samples collected before and after VIA. The sensitivity and specificity to detect CIN2+ lesions remained unaltered even when cervical samples were collected after VIA. This confirmed that acetic acid wash did not alter HC2 performance. CONCLUSIONS: Collection of samples for HC2 test is feasible immediately after VIA.