RESUMO
An important goal of synthetic biology involves the extension and standardization of novel biological elements for applications in medicine and biotechnology. Transcriptional interference, occurring in sets of convergent promoters, offers a promising mechanism for building elements for the design of tunable gene regulation. Here, we investigate the transcriptional interference mechanisms of antisense roadblock and RNA polymerase traffic in a set of convergent promoters as novel modules for synthetic biology. We show examples of elements, including antisense roadblock, relative promoter strengths, interpromoter distance, and sequence content that can be tuned to give rise to repressive as well as cooperative behaviors, therefore resulting in distinct gene expression patterns. Our approach will be useful toward engineering new biological devices and will bring new insights to naturally occurring cis-antisense systems. Therefore, we are reporting a new biological tool that can be used for synthetic biology.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regiões Promotoras Genéticas , RNA Antissenso/genética , Biologia Sintética/métodos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Análise de Sequência de DNA , Transcrição GênicaRESUMO
AMP-activated protein kinase (AMPK) is the central component of a cellular signaling system that regulates multiple metabolic enzymes and pathways in response to reduced intracellular energy levels. The transcription factor hepatic nuclear factor 4alpha (HNF4alpha) is an orphan nuclear receptor that regulates the expression of genes involved in energy metabolism in the liver, intestine, and endocrine pancreas. Inheritance of a single null allele of HNF4alpha causes diabetes in humans. Here we demonstrate that AMPK directly phosphorylates HNF4alpha and represses its transcriptional activity. AMPK-mediated phosphorylation of HNF4alpha on serine 304 had a 2-fold effect, reducing the ability of the transcription factor to form homodimers and bind DNA and increasing its degradation rate in vivo. These results demonstrate that HNF4alpha is a downstream target of AMPK and raise the possibility that one of the effects of AMPK activation is reduced expression of HNF4alpha target genes.