A profusion of upstream open reading frame mechanisms in polyamine-responsive translational regulation

In many eukaryotic mRNAs one or more short ‘upstream’ open reading frames, uORFs, precede the initiator of the main coding sequence. Upstream ORFs are functionally diverse as illustrated by their variety of features in polyamine pathway biosynthetic mRNAs. Their propensity to act as sensors for regulatory circuits and to amplify the signals likely explains their occurrence in most polyamine pathway mRNAs. The uORF-mediated polyamine responsive autoregulatory circuits found in polyamine pathway mRNAs exemplify the translationally regulated dynamic interface between components of the proteome and metabolism.     

基于细菌群感效应人工构建分子开关

对生物体内已有的或者人工组装的生物合成途径进行优化操作涉及两个重要问题:代谢途径中关键酶的活性及蛋白表达水平。对于酶表达水平的研究,传统的做法是采用强启动子控制下的靶蛋白过量表达策略。靶蛋白的过量表达通常会导致细胞内积累大量的无活性包涵体,从而严重影响细胞的生理状态和相关生物途径的有效运转。针对这一问题,设计一种分子开关来精确调控生物合成过程中关键酶的表达水平,对于研究生物合成途径的代谢节律以及促进生物合成途径高效运转都具有重要的实用价值。基于细菌群落中普遍存在群感效应的基本原理并结合酶促催化的动力学特征,首先在大肠杆菌群落中建立信号分子高丝氨酸内酯(AHL)介导的细胞–细胞交流机制,将靶基因egfp置入到启动子PluxI的控制之下。在细胞生长过程中,产生的AHL累积到一定浓度启动靶基因表达。通过在细胞生长的不同阶段启动AHL降解酶AiiA的表达控制环境中信号分子AHL的浓度水平,从而控制靶基因egfp的转录效率,最终实现对靶蛋白EGFP表达水平的精确控制。通过检测细胞的生长状态、靶基因在mRNA水平、蛋白质水平的表达情况证明人工设计的分子开关可以便捷高效地控制靶基因表达水平,具有时空调节的严谨性。该分子开关有望广泛应用于代谢工程和合成生物学等研究领域中。     

Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene

The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.     

Temporal and spatial control of expression of anthocyanin biosynthetic genes in developing flowers of Antirrhinum majus

The co-ordination of expression of anthocyanin biosynthetic genes was studied in developing flowers. Four genes encoding enzymes operating late in the anthocyanin biosynthetic pathway are induced together during flower development but the early steps appear to be induced more rapidly. Co-ordination of expression could imply a common regulatory mechanism controlling the expression of metabolically related genes. The data presented here show that while four genes may share such a mechanism for the control of their expression during flower development, different control processes regulate the early steps of the pathway. Spatially, gene expression is patterned across the flower and appears to be very similar for all the biosynthetic genes. However, the observed influence of the regulatory gene Delila shows that the spatial co-ordination of gene expression must involve more than one regulatory system. Delila itself appears to have a dual function, being required for activation of expression of the later genes in the flower tube but repressing chalcone synthase gene expression in the mesophyll of the corolla lobes. It is postulated that common signals induce the expression of genes in the pathway during flower development. The data presented here suggest that the same regulatory mechanism interprets these signals for four of the genes encoding the later biosynthetic enzymes, but that different or modified mechanisms interpret the signals to control expression of chalcone synthase and chalcone isomerase genes in Antirrhinum flowers.     

Secretory plasticity of pituitary cells: a mechanism of hormonal regulation

Pituitary somatotropes and melanotropes have enabled us to investigate the molecular basis and functional dynamics underlying secretory plasticity, an ability of endocrine cells to adapt their activity to the changing physiologic requirements, which generates discrete cell subpopulations within each cell hormonal type. Porcine somatotropes comprise two morphologically distinct subpopulations of low- (LD) and high-density (HD) cells, separable by Percoll gradient, that respond differently to hypothalamic regulators. In LD somatotropes, somatostatin (SRIF) inhibits growth hormone (GH)-releasing hormone (GHRH)-induced GH secretion. Conversely, SRIF alone stimulates GH release from HD somatotropes. These disparate SRIF actions entail a molecular signaling heterogeneity, in that SRIF increases cAMP levels in HD but not in LD cells as a requisite to stimulate GH release. GHRH-stimulated GH release also involves differential signaling in LD and HD cells: although it acts primarily through the cAMP/extracellular Ca2+ route in both somatotrope subsets, full response of LD somatotropes also requires the inositol phosphate/intracellular Ca2+ pathway. Amphibian melanotropes, which regulate skin adaptation to background color by secreting POMC-derived alpha-melanocyte-stimulating hormone (alphaMSH), also comprise two subpopulations with divergent secretory phenotypes. LD melanotropes show high biosynthetic and secretory activities and high responsiveness to multiple hypothalamic factors. Conversely, HD melanotropes constitute a hormone-storage subset poorly responsive to regulatory inputs. Interestingly, in black-adapted animals most melanotropes acquire the highly-secretory LD phenotype, whereas white-background adaptation, which requires less alphaMSH, converts melanotropes to the storage HD phenotype. These same interconversions can be reproduced in vitro using appropriate hypothalamic factors, thus revealing the pivotal role of the hypothalamus in regulating the functional dynamics of the secretory plasticity. Furthermore, this regulation likely involves a precise control of the secretory pathway, as suggested by the differential distribution in LD and HD melanotropes of key components of the intracellular transport, processing, and storage of secretory proteins. Hence, molecular signaling heterogeneity and unique secretory pathway components seem to relevantly contribute to the control of secretory plasticity, thereby enabling endocrine cells to finely adjust their dynamic response to the specific hormonal requirements.     

Interdependency of brassinosteroid and auxin signaling in Arabidopsis

How growth regulators provoke context-specific signals is a fundamental question in developmental biology. In plants, both auxin and brassinosteroids (BRs) promote cell expansion, and it was thought that they activated this process through independent mechanisms. In this work, we describe a shared auxin:BR pathway required for seedling growth. Genetic, physiological, and genomic analyses demonstrate that response from one pathway requires the function of the other, and that this interdependence does not act at the level of hormone biosynthetic control. Increased auxin levels saturate the BR-stimulated growth response and greatly reduce BR effects on gene expression. Integration of these two pathways is downstream from BES1 and Aux/IAA proteins, the last known regulatory factors acting downstream of each hormone, and is likely to occur directly on the promoters of auxin:BR target genes. We have developed a new approach to identify potential regulatory elements acting in each hormone pathway, as well as in the shared auxin:BR pathway. We show that one element highly overrepresented in the promoters of auxin- and BR-induced genes is responsive to both hormones and requires BR biosynthesis for normal expression. This work fundamentally alters our view of BR and auxin signaling and describes a powerful new approach to identify regulatory elements required for response to specific stimuli.     

Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis

How growth regulators provoke context-specific signals is a fundamental question in developmental biology. In plants, both auxin and brassinosteroids (BRs) promote cell expansion, and it was thought that they activated this process through independent mechanisms. In this work, we describe a shared auxin:BR pathway required for seedling growth. Genetic, physiological, and genomic analyses demonstrate that response from one pathway requires the function of the other, and that this interdependence does not act at the level of hormone biosynthetic control. Increased auxin levels saturate the BR-stimulated growth response and greatly reduce BR effects on gene expression. Integration of these two pathways is downstream from BES1 and Aux/IAA proteins, the last known regulatory factors acting downstream of each hormone, and is likely to occur directly on the promoters of auxin:BR target genes. We have developed a new approach to identify potential regulatory elements acting in each hormone pathway, as well as in the shared auxin:BR pathway. We show that one element highly overrepresented in the promoters of auxin- and BR-induced genes is responsive to both hormones and requires BR biosynthesis for normal expression. This work fundamentally alters our view of BR and auxin signaling and describes a powerful new approach to identify regulatory elements required for response to specific stimuli.     

DSF-家族群体感应信号生物合成途径与调控机制研究进展

群体感应是微生物间相互交流的一种重要机制。Diffusible Signaling Factor (DSF)-家族群体感应信号分子存在于多种革兰氏阴性菌中,调控细菌的致病性和适应性。本文首先介绍DSF-家族群体感应信号的结构多样性与保守性、生物合成途径和两类调控机制。DSF家族群体感应信号属于一类长链不饱和脂肪酸,碳水化合物和支链氨基酸是主要合成前体;合成途径主要包括脂肪酸合成循环和兼具脱水酶和硫酯酶活性的RpfF;在黄单胞菌和伯克氏菌中分别存在2种蛋白-蛋白互作机制调控DSF生物合成。随后,综述最新相关研究结果,提出顺式-2-十二碳烯酸(BDSF)可能是野油菜黄单胞菌侵染大白菜过程中所依赖的"活体"群体感应信号。最后,讨论和展望本领域下一步值得研究的关键科学问题。     

Sex in flies: what 'body--mind' dichotomy?

Sexual behavior in Drosophila results from interactions of multiple neural and genetic pathways. Male-specific fruitless (fruM) is a major component inducing male behaviors, but recent work indicates key roles for other sex-specific and sex-non-specific components. Notably, male-like courtship by retained (retn) mutant females reveals an intrinsic pathway for male behavior independent of fruM, while behavioral differences between males and females with equal levels of fruM expression indicate involvement of another sex-specific component. Indeed, sex-specific products of doublesex (dsxF and dsxM), that control sexual differentiation of the body, also contribute to sexual behavior and neural development of both sexes. In addition, the single product of the dissatisfaction (dsf) gene is needed for appropriate behavior in both sexes, implying additional complexities and levels of control. The genetic mechanisms controlling sexual behavior are similar to those controlling body sexual development, suggesting biological advantages of modifying an intermediate intrinsic pathway in generation of two substantially different behavioral or morphological states.     

The regulatory design of an allosteric feedback loop: the effect of saturation by pathway substrate

Aspects of metabolic regulation can be fruitfully studied with a combination of generic modelling, control analysis and graphical analysis using rate characteristics. This paper analyses a prototypical supply-demand system consisting of a biosynthetic subsystem subject to allosteric inhibition by its product and a demand process that consumes this product. The effect of changes in affinity of the committing supply enzyme for the pathway substrate on the regulatory properties of the supply subsystem is compared for the Monod-Wyman-Changeux and the reversible Hill allosteric enzyme models. We found that the Hill model has a distinct advantage in that the steady-state concentration at which it maintains the product is set by the half-saturating product concentration and is independent of changes in the degree of saturation for substrate. In contrast, with the Monod-Wyman-Changeux model this set point varies with affinity for substrate. Explicitly incorporating reversibility in all rate equations made it possible to distinguish between kinetic and thermodynamic aspects of regulation. Combining the supply and demand rate characteristics allows us to explore both the control distribution at steady state and the regulatory performance of the system over a wide range of demand activities.     

A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Gibberellin biosynthesis: its regulation by endogenous and environmental signals

The hormone gibberellin (GA) plays an essential role in many aspects of plant growth and development, such as seed germination, stem elongation and flower development. In recent years, exciting progress has been made in understanding how the biosynthesis of this hormone is regulated by endogenous and environmental factors. This has resulted from isolation of genes encoding enzymes involved in GA biosynthesis and metabolism, which also enabled us to manipulate the pathway by modifying the expression of these genes in transgenic plants. In addition, new GA response mutants provided information about how signaling components are involved in feedback regulation of the GA biosynthetic pathway.     

Gene network dynamics controlling keratinocyte migration

Translation of large‐scale data into a coherent model that allows one to simulate, predict and control cellular behavior is far from being resolved. Assuming that long‐term cellular behavior is reflected in the gene expression kinetics, we infer a dynamic gene regulatory network from time‐series measurements of DNA microarray data of hepatocyte growth factor‐induced migration of primary human keratinocytes. Transferring the obtained interactions to the level of signaling pathways, we predict in silico and verify in vitro the necessary and sufficient time‐ordered events that control migration. We show that pulse‐like activation of the proto‐oncogene receptor Met triggers a responsive state, whereas time sequential activation of EGF‐R is required to initiate and maintain migration. Context information for enhancing, delaying or stopping migration is provided by the activity of the protein kinase A signaling pathway. Our study reveals the complex orchestration of multiple pathways controlling cell migration.     

Adenosine diphosphate glucose pyrophosphorylase, a rate‐limiting step in starch biosynthesis

Starch represents the major component of virtually all plant‐derived foods consumed by man and animal. Hence, a thorough understanding of the starch biosynthetic pathway is critically important not only in understanding the biosynthesis of a major plant storage product, but also in allowing the genetic manipulation of both starch quality and quantity for human benefit. A major goal in these studies has been the identification of key steps in controlling starch levels. Evidence from a number of independent approaches clearly points to the enzyme adenosine diphosphate glucose pyrophosphorylase (AGPase) as a key regulatory step in starch synthesis. Here we highlight and summarize our understanding of this important enzyme.     

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.