A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co‐expressed to function. The DNA‐binding loop is encoded in a C‐terminal 285‐aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601‐aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67‐aa N‐terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.United States. Office of Naval Research (N00014‐13‐1‐0074)National Institutes of Health (U.S.) (5R01GM095765)National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center (SA5284‐11210))United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship (NDSEG) Program))Hertz Foundation (Fellowship

An RxLR effector from phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus

The plant immune system is activated following the perception of exposed, essential and invariant microbial molecules that are recognised as non-self. A major component of plant immunity is the transcriptional induction of genes involved in a wide array of defence responses. In turn, adapted pathogens deliver effector proteins that act either inside or outside plant cells to manipulate host processes, often through their direct action on plant protein targets. To date, few effectors have been shown to directly manipulate transcriptional regulators of plant defence. Moreover, little is known generally about the modes of action of effectors from filamentous (fungal and oomycete) plant pathogens. We describe an effector, called Pi03192, from the late blight pathogen Phytophthora infestans, which interacts with a pair of host transcription factors at the endoplasmic reticulum (ER) inside plant cells. We show that these transcription factors are released from the ER to enter the nucleus, following pathogen perception, and are important in restricting disease. Pi03192 prevents the plant transcription factors from accumulating in the host nucleus, revealing a novel means of enhancing host susceptibility

Evidence for transcription attenuation rendering cryptic a sigmaS- dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the σ S- and σ 70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by σ S RNA polymerase in vitro and that a 22-bp deletion from +63 to +84 (relative to the start site of transcription) confers σ S-dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to +60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to +85 is able to do so only from the latter. Our results suggest that the σ S-controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

The TyrR protein of Escherichia coli is a class I transcription activator

The purified TyrR protein and phenylalanine were sufficient to activate in vitro transcription from the tyrP promoter by wild-type RNA polymerase. Such TyrR-mediated activation did not occur when the mutant alpha 235 RNA polymerase was used, indicating that TyrR is a class I transcription activator.</jats:p

Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments.

We report a technique to selectively and continuously label the proteomes of individual cell types in coculture, named cell type-specific labeling using amino acid precursors (CTAP). Through transgenic expression of exogenous amino acid biosynthesis enzymes, vertebrate cells overcome their dependence on supplemented essential amino acids and can be selectively labeled through metabolic incorporation of amino acids produced from heavy isotope-labeled precursors. When testing CTAP in several human and mouse cell lines, we could differentially label the proteomes of distinct cell populations in coculture and determine the relative expression of proteins by quantitative mass spectrometry. In addition, using CTAP we identified the cell of origin of extracellular proteins secreted from cells in coculture. We believe that this method, which allows linking of proteins to their cell source, will be useful in studies of cell-cell communication and potentially for discovery of biomarkers

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

Effects of H-NS and potassium glutamate on &#963;<SUP>S</SUP>- and &#963;<SUP>70</SUP>-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the &#963;s and &#963;70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of &#963;s selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS

Toward quantitative proteomics of organ substructures: implications for renal physiology

Organs are complex structures that consist of multiple tissues with different levels of gene expression. To achieve comprehensive coverage and accurate quantitation data, organs ideally should be separated into morphologic and/or functional substructures before gene or protein expression analysis. However, because of complex morphology and elaborate isolation protocols, to date this often has been difficult to achieve. Kidneys are organs in which functional and morphologic subdivision is especially important. Each subunit of the kidney, the nephron, consists of more than 10 subsegments with distinct morphologic and functional characteristics. For a full understanding of kidney physiology, global gene and protein expression analyses have to be performed at the level of the nephron subsegments; however, such studies have been extremely rare to date. Here we describe the latest approaches in quantitative high-accuracy mass spectrometry-based proteomics and their application to quantitative proteomics studies of the whole kidney and nephron subsegments, both in human beings and in animal models. We compare these studies with similar studies performed on other organ substructures. We argue that the newest technologies used for preparation, processing, and measurement of small amounts of starting material are finally enabling global and subsegment-specific quantitative measurement of protein levels in the kidney and other organs. These new technologies and approaches are making a decisive impact on our understanding of the (patho)physiological processes at the molecular level