Prokaryotic Gene Regulation

Improved ecdysone receptor-based inducible gene regulation system Subba R. Palli 1 , Mariana Z. Kapitskaya 2 , Mohan B. Kumar 2 and Dean E. Cress 2 1 Department of Entomology, College of Agriculture, University of Kentucky, KY, USA; 2 RHeoGene LLC, Spring House, PA, USA To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus muscu- lus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn- off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively. Keywords: gene switch; ponasterone A; receptors; EcR; RXR. Twenty hydroxyecdysone (20E) is a steroid hormone that regulates molting, metamorphosis, reproduction and vari- ous other developmental processes in insects. Ecdysone functions through a heterodimeric receptor complex com- prised of ecdysone receptor (EcR) and ultraspiracle (USP). Both EcR and USP cDNAs have been cloned from Drosophila melanogaster and several other insects [1] and were shown to be members of the steroid hormone receptor superfamily. Members of this superfamily are characterized by the presence of five modular domains, A/B (transacti- vation), C (DNA binding/heterodimerization), D (hinge, heterodimerization), E (ligand binding, heterodimerization, transactivation) and F (transactivation). Crystallographic studies on the E domain structures of several nuclear receptors showed a conserved fold composed of 11 helices (H1 and H3–H12) and two short strands (s1 and s2) [2]. Recently, the crystal structure of USP was solved by two groups [3,4], both structures showed a long H1-H3 loop and an insert between H5 and H6. These structures appear to lock USP in an inactive conformation by displacing helix 12 from agonist conformation. In both crystal structures USP had a large hydrophobic cavity, which contained phos- pholipid ligands. The crystal structure of the EcR has yet to be determined; however, homology models for CtEcR (Chironomus tentans EcR) [5], and CfEcR (Choristoneura fumiferana EcR) [6] have been generated [7,8]. Ecdysone receptors are found in insects and other related invertebrates [1]. Ecdysteroids and related compounds have been identified in plants, insects and other related inverte- brates. EcR and its ligands are not detected in vertebrates such as humans, therefore they are very good candidates for developing gene regulation systems for use in vertebrates. Insect EcR can heterodimerize with retinoid X receptor (RXR) and transactivate genes that are placed under the control of ecdysone response elements (EcRE) in various cellular backgrounds including mammalian cells. The EcR- based gene switch is being developed for use in various applications including gene therapy, expression of toxic proteins in cell lines as well as for cell-based drug discovery assays [9–17]. After initial reports [18,19] on the function of EcR as an ecdysteroid dependent transcription factor in cultured mammalian cells, No et al. [20] used D. melanogaster EcR (DmEcR) and human RXRa to develop Prokaryotic Gene Regulation Prokaryotic Gene Regulation Bởi: OpenStaxCollege The DNA of prokaryotes is organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers Repressors are proteins that suppress transcription of a gene in response to an external stimulus, whereas activators are proteins that increase the transcription of a gene in response to an external stimulus Finally, inducers are small molecules that either activate or repress transcription depending on the needs of the cell and the availability of substrate The trp Operon: A Repressor Operon Bacteria such as E coli need amino acids to survive Tryptophan is one such amino acid that E coli can ingest from the environment E coli can also synthesize tryptophan using enzymes that are encoded by five genes These five genes are next to each other in what is called the tryptophan (trp) operon ([link]) If tryptophan is present in the environment, then E coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized 1/7 Prokaryotic Gene Regulation The five genes that are needed to synthesize tryptophan in E coli are located next to each other in the trp operon When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence This physically blocks the RNA polymerase from transcribing the tryptophan genes When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed A DNA sequence that codes for proteins is referred to as the coding region The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon Just before the coding region is the transcriptional start site This is the region of DNA to which RNA polymerase binds to initiate transcription The promoter sequence is upstream of the transcriptional start site; each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription A DNA sequence called the operator sequence is encoded between the promoter region and the first trp coding gene This operator contains the DNA code to which the repressor protein can bind When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators Link to Learning 2/7 Prokaryotic Gene Regulation Watch this video to learn more about the trp operon Catabolite Activator Protein (CAP): An Activator Regulator Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them For example, when glucose is scarce, E coli bacteria can turn to other sugar sources for fuel To this, new genes to process these alternate genes must be transcribed When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E coli When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources ([link]) In these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes When glucose levels fall, E coli may use other sugars for fuel but must transcribe new genes to so As glucose supplies become limited, cAMP levels increase This cAMP binds to ...Characterization of the Drosophila Methoprene -tolerant gene product Juvenile hormone binding and ligand-dependent gene regulation Ken Miura, Masahito Oda, Sumiko Makita and Yasuo Chinzei Department of Medical Zoology, School of Medicine, Mie University, Tsu City, Japan Insect development and reproduction are regulated by two classes of lipid-soluble hormones, the ecdysteroids and juvenile hormones (JHs). The ecdysteroids activate target genes through a heterodimeric receptor complex composing the ecdysone receptor and ultraspiracle (USP) proteins, both of which are members of the nuc- lear steroid ⁄ thyroid ⁄ retinoid receptor superfamily [1]. During insect development, ecdysteroids induce molting while JH determines the nature of each molt by modu- lating the ecdysteroid-induced gene expression cascade [2–4]. In addition, in adult insects, JH has a wide variety of actions related to reproduction, including oogenesis, migratory behaviour and diapause [2,5,6]. The mode of molecular action of JH, however, is still obscure [7]. JHs are a family of esterified sesquiterpe- noids, whose lipid-soluble nature has suggested action directly on the genome through nuclear receptors such as ecdysteroids and the vertebrate steroid ⁄ thyroid ⁄ reti- noid hormones [5,8] although actions of JH through the cell membrane are also documented [9,10]. Many attempts have been made to identify nuclear JH receptors. Jones and Sharp [11] showed that JH III binds to the Drosophila USP protein, which is a homo- logue of the vertebrate retinoid X receptor, promoting Keywords juvenile hormone; juvenile hormone receptor; Methoprene-tolerant; Drosophila; transcription factor Correspondence K. Miura, Department of Medical Zoology, School of Medicine, Mie University, Edobashi 2-174, Tsu514-8507, Japan Fax: +81 59 231 5215 Tel: +81 59 231 5013 E-mail: k-miura@doc.medic.mie-u.ac.jp (Received 27 October 2004, revised 20 December 2004, accepted 4 January 2005) doi:10.1111/j.1742-4658.2005.04552.x Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene- tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. We have now examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regu- lation. In vitro synthesized MET directly bound to JH III with high affinity (K d ¼ 5.3 ± 1.5 nm, mean ± SD), consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA- dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III  JH II > JH I > methoprene; compounds which are only structur- ally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent tran- scription factor. Abbreviations Ahr, aryl hydrocarbon receptor; Arnt, Ahr nuclear translocator; bHLH, basic helix-loop-helix; DBD, DNA binding domain; DCC, dextran-coated charcoal; EGFP, enhanced green REVIEW ARTICLE Functional interplay between viral and cellular SR proteins in control of post-transcriptional gene regulation Ming-Chih Lai 1, *, Tsui-Yi Peng 1,2, * and Woan-Yuh Tarn 1 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2 Institute of Molecular Medicine, National Tsing Hua University, Hsin-Chu, Taiwan Introduction Arginine ⁄ serine (RS) dipeptide repeats are present in a number of cellular proteins, termed SR proteins, that primarily participate in nuclear precursor (pre)-mRNA splicing [1–3]. RS domain variants, such as serine and arginine-rich motifs or arginine–aspartate or arginine– glutamate dipeptide-rich domains, are also found in many nuclear proteins. In addition to the RS domains, SR splicing factors often contain one or more RNA recognition motifs. SR proteins function in both constitutive and regulated splicing via binding to cis-elements of pre-mRNA or interaction with other splicing factors. The RS domain interacts with both proteins and RNAs [1–3]. In particular, intermolecular interactions between SR proteins, which are important for spliceosome assembly and splice site determination during pre-mRNA splicing, are mediated by their RS domains [3]. The RS domain also acts as a nuclear localization signal and targets SR proteins to nuclear speckled domains, where splicing factors are concen- trated, for storage [1]. An important biochemical property of the RS domain is its differential phosphorylation at multiple serine and threonine residues. The RS domain is primarily phos- phorylated by SR protein-specific kinases (SRPKs), and Keywords Alternative splicing; kinases; phosphatases; phosphorylation; post-transcriptional control; pre-mRNA splicing; RS domain; SR proteins; viral problems; virus Correspondence W Y. Tarn, Institute of Biomedical Sciences, Academia Sinica, 128 Academy Road, Section 2, Nankang, Taipei 11529, Taiwan Fax: +886 2 2782 9142 Tel: +886 2 2652 3052 E-mail: wtarn@ibms.sinica.edu.tw *These authors contributed equally to this work (Received 3 November 2008, revised 14 December 2008, accepted 9 January 2009) doi:10.1111/j.1742-4658.2009.06894.x Viruses take advantage of cellular machineries to facilitate their gene expression in the host. SR proteins, a superfamily of cellular precursor mRNA splicing factors, contain a domain consisting of repetitive argi- nine ⁄ serine dipeptides, termed the RS domain. The authentic RS domain or variants can also be found in some virus-encoded proteins. Viral pro- teins may act through their own RS domain or through interaction with cellular SR proteins to facilitate viral gene expression. Numerous lines of evidence indicate that cellular SR proteins are important for regulation of viral RNA splicing and participate in other steps of post-transcriptional viral gene expression control. Moreover, viral infection may alter the expression levels or modify the phosphorylation status of cellular SR proteins and thus perturb cellular precursor mRNA splicing. We review our current understanding of the interplay between virus and host in post-transcriptional regulation of gene expression via RS domain-containing proteins. Abbreviations CTE, constitutive transport element; E4, early region 4; EV, epidermodysplasia verruciformis; HBV, hepatitis B virus; HCV, hepatitis C virus; hnRNP, heterogeneous nuclear ribonucleoprotein; HPV, human papillomavirus; HSV, herpes simplex virus; IRES, internal ribosome entry site; N, nucleocapsid; PP, protein phosphatase; REVIEW ARTICLE Gene regulation by tetracyclines Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes Christian Berens and Wolfgang Hillen Lehrstuhl fu ¨ r Mikrobiologie, Institut fu ¨ r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universita ¨ t Erlangen-Nu ¨ rnberg; Germany The Tet repressor protein (TetR) regulates transcription of a family of tetracycline (tc) resistance determinants in Gram-negative bacteria. The resistance protein TetA, a membrane-spanning H + -[tcÆM] + antiporter, must be sen- sitively regulated because its expression is harmful in the absence of tc, yet it has to be expressed before the drugs’ concentration reaches cytoplasmic levels inhibitory for protein synthesis. Consequently, TetR shows highly speci- fic tetO binding to reduce basal expression and high affinity to tc to ensure sensitive induction. Tc can cross biological membranes by diffusion enabling this inducer to penetrate the majority of cells. These regulatory and pharmacological properties are the basis for application of TetR to selec- tively control the expression of single genes in lower and higher eukaryotes. TetR can be used for that purpose in some organisms without further modifications. In mam- mals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator. Mechanistic understanding and the ability to engineer and screen for mutants with specific properties allow tailoring of the DNA recognition specificity, the response to inducer tc and the dimerization specificity of TetR-based eukary- otic regulators. This review provides an overview of the TetR properties as they evolved in bacteria, the functional modifications necessary to transform it into a convenient, specific and efficient regulator for use in eukaryotes and how the interplay between structure ) function studies in bacteria and specific requirements of particular applica- tions in eukaryotes have made it a versatile and highly adaptable regulatory system. Keywords: antibiotic resistance; disease models; fusion pro- tein; inducible gene expression; ligand-binding specificity; mammalian cell lines; protein engineering; structure–activity relationship; Tet repressor; transgenic organism. Properties of bacterial Tet systems Efflux-mediated tetracycline resistance is always regulated in Gram-negative bacteria In Gram-negative bacteria, resistance to tetracyclines (tc) is mediated by the TetA protein, a proton-[tcÆMg] + anti- porter embedded in the cytoplasmic membrane [1,2]. Eleven tc resistance determinants (Tet classes A–E, G, H, J, Z, 30, and 33 [3–5]) share the organization of structural and regulatory genes (reviewed in [6]). In enteric bacteria, the efflux-encoding tetA genes are strictly regulated at the level of transcription by the tc-responsive Tet repressor (TetR). In the absence of inducer, TetR dimers bind to the operators tetO 1 and tetO 2 , shutting down transcription of its own gene, tetR, and of the resistance gene, tetA.Oncetchas entered the cell, it binds TetR with high affinity as a [tcÆMg] + complex [7]. This induces a conformational change in TetR [8] resulting in dissociation from tetO [9]. The following expression burst of TetA and TetR leads to a rapid reduction of the cytoplasmic tc concentration [10] which, in turn, shuts expression of both genes off again. Expression MINIREVIEW Collective behavior in gene regulation: Metabolic clocks and cross-talking Michele M. Bianchi Department of Cell and Developmental Biology, University of Rome ‘La Sapienza’, Italy By cosmic rule, as day yields night, so winter sum- mer, war peace, plenty famine. All things change… the harmonious structure of the world depends upon opposite tensions. (Heraclitus, 500 bc) In the modern age, life scientists subscribe to the ergo- dic cell hypothesis (Fig. 1): they use homogenized tissues or cultured cells, analyze extracts and draw conclusions about a hypothetical representative cell on the basis that all cells are ‘on average’ identical over (short) time and space scales [1]. In this representation (statistical mechanics, where it allowed a microscopic basis to be given to thermodynamics), the average of a process parameter for a single cell over time and the average over the statistical ensemble of individuals at a given time coincide. In the ergodic hypothesis, genes are generally divided into housekeeping genes, which are always expressed, and regulated genes, which are expressed or repressed under the effect of external signals. The external signal might have various origins: an environ- mental condition, a physiological signal from other regions of a multicellular organism, the result of a developmental programme, epigenetic control and so on. In any case, these external signals occur inciden- tally and ‘on average’ elicit the same response in all cells; this means that they may have different effects depending on the status of each cell but, given that the population is very large and a point in time displays the same distribution of states, the average result is the same irrespective of time. If we want to study the behavior of a single cell in a time-dependent manner, by analysing a representative population of individuals, we must artificially put all the cells into the same state by synchronization, in order to collapse the ensemble distribution into a single state. This collapse is usually unstable and, after a relatively short time, the cell pop- ulation reverts to the statistical distribution of states. Keywords circadian clock; cross-talk; cycles; ergodic system; message; metabolism; redox; synchronization; transcription dynamics; ultradian clock Correspondence M. M. Bianchi, Department of Cell and Developmental Biology, p.le Aldo Moro 5, 00185 Rome, Italy Fax: +39 064 991 2351 Tel: +39 064 991 2215 E-mail: michele.bianchi@uniroma1.it (Received 10 December 2007, accepted 30 January 2008) doi:10.1111/j.1742-4658.2008.06397.x Biological functions governed by the circadian clock are the evident result of the entrainment operated by the earth’s day and night cycle on living organisms. However, the circadian clock is not unique, and cells and organisms possess many other cyclic activities. These activities are difficult to observe if carried out by single cells and the cells are not coordinated but, if they can be detected, cell-to-cell cross-talk and synchronization among cells must exist. Some of these cycles are metabolic and cell syn- chronization is due to small molecules acting as metabolic messengers. We propose a short survey of cellular cycles, paying special attention to meta- bolic cycles and cellular cross-talking, particularly when the synchroniza- tion of metabolism or, more generally, cellular functions are concerned. Questions arising from the observation of phenomena based on cell com- munication and from basic cellular cycles are also proposed. Abbreviations ROS, reactive oxygen species; YGO, yeast glycolytic oscillation; YMC, yeast metabolic cycle. 2356 FEBS Journal 275 (2008) 2356–2363 ª 2008 The Author Journal compilation ª 2008 FEBS Clocks Looking closer at the cell or organism and ... region upstream of the genes required to use other sugar sources 3/7 Prokaryotic Gene Regulation The lac Operon: An Inducer Operon The third type of gene regulation in prokaryotic cells occurs... the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators Link to Learning 2/7 Prokaryotic Gene Regulation. . .Prokaryotic Gene Regulation The five genes that are needed to synthesize tryptophan in E coli are located next to