Specific Transcription Initiation Research Articles

IPro2L-PSTKNC: A Two-Layer Predictor for Discovering Various Types of Promoters by Position Specific of Nucleotide Composition

Promoters are DNA regulatory elements located proximal to the transcription start site, which are in charge of the initiation of specific gene transcription. In Escherichia coli, promoters can be recognized by σ factors that have multiple families based on distinct function and structure, such as σ24, σ28, σ32, σ38, σ54 and σ70. At present, biological methods are mainly used to identify these promoters. However, because it is time-consuming and material-consuming to do biological experiments, computational biology algorithm has emerged as a more effective way to predict the classification. In this study, we develop a novel two-layer seamless predictor called iPro2L-PSTKNC to identify the promoters of the E. coli genome, which based on the feature extraction model we newly proposed that is named as the position specific tendencies of k-mer nucleotide composition (PSTKNC). On the first layer, it is a binary classification predicting whether a sequence is promoter or not. And the second layer is a multiple classification identifying which type the identified promoter belongs to. The ensemble classification SVM performsbest comparing with other algorithms, which gets a promising accuracy and the Matthews correlation coefficient (MCC) at [Formula: see text] and [Formula: see text]. Our data and code are available at https://github.com/lyuyinuo/iPro2L-PSTKNC.

A simple protocol to purify human TFIID free of the MED26 subunit of mediator complex

The general transcription factor TFIID is a multiprotein complex that is essential for specific transcription initiation by RNA polymerase II. It is composed of the TATA box-binding protein (TBP) and ~13 different TBP-associated factors (TAFs). Purification of TFIID free of other general transcription factors and coactivators is essential to analyze the transcription regulatory mechanisms in reconstituted systems in vitro. A breakthrough in TFIID purification was the generation of HeLa cell lines that express a FLAG epitope-tagged TBP subunit and immunopurification protocols with monoclonal anti-FLAG antibodies. Purification of TFIID from HeLa nuclear extracts generally required a two-step purification procedure involving phosphocellulose P11 chromatography followed by anti-flag M2 affinity purification (Chiang et al., 1993; Ge et al., 1996) [1,2]. Here we show first that the MED26 (CRSP70) coactivator subunit of Mediator co-purifies with TFIID in the above two-step protocol and interacts strongly with TFIID under high salt conditions. We further show that a MED26-free TFIID complex can be obtained by including a simple additional DE52 chromatography step following P11 fractionation. Thus, we demonstrate that MED26 strongly interacts with TFIID and recommend the use of a P11-DE52-M2 resin affinity three-step purification procedure to obtain MED26-free TFIID for analyzing Mediator-dependent transcription regulatory mechanisms in purified transcription systems in vitro.

A non-canonical multisubunit RNA polymerase encoded by the AR9 phage recognizes the template strand of its uracil-containing promoters.

AR9 is a giant Bacillus subtilis phage whose uracil-containing double-stranded DNA genome encodes distant homologs of β and β’ subunits of bacterial RNA polymerase (RNAP). The products of these genes are thought to assemble into two non-canonical multisubunit RNAPs - a virion RNAP (vRNAP) that is injected into the host along with phage DNA to transcribe early phage genes, and a non-virion RNAP (nvRNAP), which is synthesized during the infection and transcribes late phage genes. We purified the AR9 nvRNAP from infected B. subtilis cells and characterized its transcription activity in vitro. The AR9 nvRNAP requires uracils rather than thymines at specific conserved positions of late viral promoters. Uniquely, the nvRNAP recognizes the template strand of its promoters and is capable of specific initiation of transcription from both double- and single-stranded DNA. While the AR9 nvRNAP does not contain homologs of bacterial RNAP α subunits, it contains, in addition to the β and β’-like subunits, a phage protein gp226. The AR9 nvRNAP lacking gp226 is catalytically active but unable to bind to promoter DNA. Thus, gp226 is required for promoter recognition by the AR9 nvRNAP and may represent a new group of transcription initiation factors.

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

HPr antagonizes the anti-σ 70 activity of Rsd in Escherichia coli

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

Anti-Sigma Factors in E. coli: Common Regulatory Mechanisms Controlling Sigma Factors Availability

In bacteria, transcriptional regulation is a key step in cellular gene expression. All bacteria contain a core RNA polymerase that is catalytically competent but requires an additional σ factor for specific promoter recognition and correct transcriptional initiation. The RNAP core is not able to selectively bind to a given σ factor. In contrast, different σ factors have different affinities for the RNAP core. As a consequence, the concentration of alternate σ factors requires strict regulation in order to properly control the delicate interplay among them, which favors the competence for the RNAP core. This control is archived by different σ/anti-σ controlling mechanisms that shape complex regulatory networks and cascades, and enable the response to sudden environmental cues, whose global understanding is a current challenge for systems biology. Although there have been a number of excellent studies on each of these σ/anti-σ post-transcriptional regulatory systems, no comprehensive comparison of these mechanisms in a single model organism has been conducted. Here, we survey all these systems in E. coli dissecting and analyzing their inner workings and highlightin their differences. Then, following an integral approach, we identify their commonalities and outline some of the principles exploited by the cell to effectively and globally reprogram the transcriptional machinery. These principles provide guidelines for developing biological synthetic circuits enabling an efficient and robust response to sudden stimuli.

Abstract 5117: The expression of ERG oncoprotein and Annexin A2 show a strong inverse correlation in prostate cancers.

Abstract Introduction: Gene fusion of the TMPRSS2 promoter and ERG that gives rise to aberrant ERG expression is detected in over 50% of prostate cancers (CaP). ERG overexpression abrogates cell differentiation, promotes cell invasion and epithelial to mesenchymal transition, and additional Pten loss or AKT up-regulation enhances the transformation into well-differentiated prostate adenocarcinoma. Annexin A2 (ANXA2) is a calcium–dependent phospholipid–binding protein that regulates cell adhesion, cellular junction formation, actin remodeling and genesis of cell polarity. Evidence from a variety of malignancies suggests that ANXA2 is vital for promoting adhesion, migration and metastasis in cancer cells. In CaP, ANXA2 expression is lost or reduced, however, is increased in high grade tumors. Microarray gene expression studies have shown an inverse correlated ERG and ANXA2 expression. We examined the cancer associated functions of ERG and ANXA2 such as cell invasion, cell migration and cell polarity in CaP cell lines. Methods: ANXA2 and ERG cellular localization were monitored using fluorescence microscopy. The prostate specific transcriptional initiation site of ANXA2 was defined by 5’–RACE. Ch-IP Assay was used to show the recruitment of ERG to the ANXA2 promoter. Cases consisting of well differentiated and poorly differentiated CaP with and without TMPRSS2-ERG fusion were stained with ANXA2 and ERG antibodies to determine correlation in expression. Results: The inverse correlation between ERG and ANXA2 protein expression was confirmed by ERG knockdown experiments in VCaP cells. ERG overexpression in prostate adenocarcinoma is correlated with the loss ANXA2 expression in 90% of cases examined. A subset of ERG positive tumors exhibit small clusters of cells with clonal expression of ANXA2. We evaluated the patterns of ERG-ANXA2 expression against available clinical-pathological features. Conclusions: Our findings suggest that the expression of ANXA2 and its function in establishing cell polarity and cellular junctions may be disrupted by ERG overexpression through the transcriptional repression of ANXA2. We hypothesize that the reemergence of ANXA2 expression in advanced tumors reflects the accumulation of genetic hits that resulted in a more aggressive tumor with higher metastatic capacity. An IHC based assay with combined ERG and ANXA2 antibody cocktail could enhance the pathological evaluation of prostate tumors discern the re-expression of ANXA2 in ERG positive tumors and improve the prognosis and treatment of CaPs. This research was in part supported by the National Cancer Institute R01CA162383 grant to S. S. and USU–CPDR funds. Citation Format: Nick E. Griner, Shyh-Han Tan, Denise Young, Albert Dobi, Gyorgy Petrovics, Isabell Sesterhenn, Shiv Srivastava. The expression of ERG oncoprotein and Annexin A2 show a strong inverse correlation in prostate cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5117. doi:10.1158/1538-7445.AM2013-5117

Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors

BackgroundThe various steps of mRNP biogenesis (transcription, processing and export) are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation.ResultsWe investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC) formation.ConclusionsOur results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

TAF‐dependent basal transcription function of new Cofactors through the Initiator element

Specific transcription initiation by eukaryotic RNA polymerase II is controlled by core promoter DNA elements that recruit general/basal transcription factors (GTFs) and by distal promoter and enhancer sequences that bind regulatory factors. The TATA box (at −30) and the Initiator (INR) at the transcription start site (+1) are core promoter elements that are variably present in eukaryotic promoters, and are bound, respectively, by the TBP and TAF components of the TFIID complex. TATA and INR elements synergize to enhance TFIID binding and basal transcription and their presence or absence influences the activation of core promoters by specific regulators and enhancers. While the INR is recognized by the TAF1 and TAF2 subunits of TFIID, INR‐dependent transcription and TATAINR synergy are not observed in purified transcription systems reconstituted with TFIID, the other GTFs (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH) and RNA polymerase II, but requires additional TAFs and Initiator‐dependent cofactors (TICs) that have remained elusive. Here, we report the identification of several of these new Cofactors that potentiate the basal transcription function of TFIID/TAFs through the INR element and allow TATA‐INR synergy.

TAF4b and Jun/Activating Protein-1 Collaborate to Regulate the Expression of Integrin α6 and Cancer Cell Migration Properties

The TAF4b subunit of the transcription factor IID, which has a central role in transcription by polymerase II, is involved in promoter recognition by selective recruitment of activators. The activating protein-1 (AP-1) family members participate in oncogenic transformation via gene regulation. Utilizing immunoprecipitation of endogenous protein complexes, we documented specific interactions between Jun family members and TATA box binding protein-associated factors (TAF) in colon HT29 adenocarcinoma cells. Particularly, TAF4b and c-Jun were found to colocalize and interact in the nucleus of advanced carcinoma cells and in cells with epithelial-to-mesenchymal transition (EMT) characteristics. TAF4b was found to specifically regulate the AP-1 target gene involved in EMT integrin alpha6, thus altering related cellular properties such as migration potential. Using a chromatin immunoprecipitation approach in colon adenocarcinoma cell lines, we further identified a synergistic role for TAF4b and c-Jun and other AP-1 family members on the promoter of integrin alpha6, underlining the existence of a specific mechanism related to gene expression control. We show evidence for the first time of an interdependence of TAF4b and AP-1 family members in cell type-specific promoter recognition and initiation of transcription in the context of cancer progression and EMT.

Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression

The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.

Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids

We have investigated the function of one of the six plastid sigma-like transcription factors, sigma 3 (SIG3), by analysing two different Arabidopsis T-DNA insertion lines having disrupted SIG3 genes. Hybridization of wild-type and sig3 plant RNA to a plastid specific microarray revealed a strong reduction of the plastid psbN mRNA. The microarray result has been confirmed by northern blot analysis. The SIG3-specific promoter region has been localized on the DNA by primer extension and mRNA capping experiments. Results suggest tight regulation of psbN gene expression by a SIG3-PEP holoenzyme. The psbN gene is localized on the opposite strand of the psbB operon, between the psbT and psbH genes, and the SIG3-dependent psbN transcription produces antisense RNA to the psbT–psbH intergenic region. We show that this antisense RNA is not limited to the intergenic region, i.e. it does not terminate at the end of the psbN gene but extends as antisense transcript to cover the whole psbT coding region. Thus, by specific transcription initiation at the psbN gene promoter, SIG3-PEP holoenzyme could also influence the expression of the psbB operon by producing psbT antisense RNA.

Growth Phase-Dependent Regulation of the Extracytoplasmic Stress Factor, σ E , by Guanosine 3′,5′-Bispyrophosphate (ppGpp)

The sigma subunit of procaryotic RNA polymerases is responsible for specific promoter recognition and transcription initiation. In addition to the major sigma factor, sigma 70, in Escherichia coli, which directs most of the transcription in the cell, bacteria possess multiple, alternative sigma factors that direct RNA polymerase to distinct sets of promoters in response to environmental signals. By activating an alternative sigma factor, gene expression can be rapidly reprogrammed to meet the needs of the cell as the environment changes. Sigma factors are subject to multiple levels of regulation that control their levels and activities. The alternative sigma factor sigmaE in Escherichia coli is induced in response to extracytoplasmic stress. Here we demonstrate that sigmaE can also respond to signals other than extracytoplasmic stress. sigmaE activity increases in a growth phase-dependent manner as a culture enters stationary phase. The signaling pathway that activates sigmaE during entry into stationary phase is dependent upon the alarmone guanosine 3',5'-bispyrophosphate (ppGpp) and is distinct from the pathway that signals extracytoplasmic stress. ppGpp is the first cytoplasmic factor shown to control sigmaE activity, demonstrating that sigmaE can respond to internal signals as well as signals originating in the cell envelope. ppGpp is a general signal of starvation stress and is also required for activation of the sigmaS and sigma 54 alternative sigma factors upon entry into stationary phase, suggesting that this is a key mechanism by which alternative sigma factors can be activated in concert to provide a coordinated response to nutritional stress.

Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.

Photoactivatable Analogues of the Initiating Substrates of RNA Polymerase II Based on Aryl Azide Derivatives of NTP γ-Amidophosphate: Synthesis and Chemical and Photochemical Reactions of Functional Groups

Photoactivatable derivatives Ar-NH-(CH2)n-NHpppB (where Ar = p-azidophenyl (A1), 5-azido-2-nitrobenzoyl (A2), or 4-azido-2,3,5,6-tetrafluorobenzoyl (A3) group; B = Ado or Guo; n = 2, 3, or 4) were synthesized. The phosphoroamidate bond stability was found to depend on the structure of both the heterocyclic and the photoactivatable groups. The derivative with A3, Ado, and n=3 is hydrolyzed with regeneration of aryl azide and ATP, whereas the other derivatives are stable in aqueous solutions. The photoanalogues with A1 and A2, B = Ado, and n = 2 or 4 were found to behave as initiating substrates toward the RNA polymerase II from Saccharomyces cerevisiae under the conditions of specific transcription initiation and control of the adenovirus late promoter. The photolysis of N-(4-azidophenyl)-1,4-diaminobutane and N-(5-azido-2-nitrobenzoyl)-1,3-diaminopropane, two functional fragments of the photoaffinity reagents, in aqueous solutions was established to result in the formation of p-benzoquinone diimine and p-nitro-N-arylhydroxylamine derivatives, respectively. The arylhydroxylamine derivatives undergo a number of transformations in aqueous solution leading to nitroso derivatives. We concluded that it is these nitroso derivatives (products of nitrene transformation, rather than the nitrene itself) that may modify proteins with reagents containing p-nitrophenylazide fragment.

Structure, expression and regulation of the cannabinoid receptor gene (CB1) in Huntington's disease transgenic mice

Loss of cannabinoid receptors (CB1) occurs prior to neurodegeneration in Huntington's disease (HD). The levels and distribution of CB1 RNA were equivalent in 3-week-old mice regardless of genotype demonstrating that the specific factors and appropriate chromatin structure that lead to the transcription of CB1 were present in the striatum of young R6/2 and R6/1 transgenic HD mice. The expression of the mutant HD transgene led progressively to decreased steady-state levels of CB1 mRNA in neurons of the lateral striatum, which was dependent on the size of the CAG repeat and relative expression of the gene encoding mutant huntingtin (HD). Although it is known that the coding region of CB1 is contained within a single exon in mice, rats and humans, the 5'-untranslated region of the mouse gene remained to be defined. CB1 mRNA is encoded by two exons separated by an 18.4-kb intron. Transcription of CB1 occurred at multiple sites within a GC-rich promoter region upstream of exon 1 encoding the 5'-UTR of CB1. There was no difference in the selection of specific transcription initiation sites associated with higher levels of CB1 expression in the striatum compared to the cortex or between the striata of wild-type and HD transgenic mice. The progressive decline in CB1 mRNA levels in R6 compared to wild-type mice was due to decreased transcription, which is consistent with the hypothesis that mutant huntingtin exerts its effects by altering transcription factor activity. The cell-specific conditions that allow for increased transcription of CB1 in the lateral striatum compared to other forebrain regions from all transcription start sites were affected by the expression of mutant huntingtin in a time-dependent manner.

Human mitochondrial transcription factor B1 interacts with the C-terminal activation region of h-mtTFA and stimulates transcription independently of its RNA methyltransferase activity.

A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated.

Vias de Sinalização da Insulina

A insulina é um hormônio anabólico com efeitos metabólicos potentes. Os eventos que ocorrem após a ligação da insulina são específicos e estritamente regulados. Definir as etapas que levam à especificidade deste sinal representa um desafio para as pesquisas bioquímicas, todavia podem resultar no desenvolvimento de novas abordagens terapêuticas para pacientes que sofrem de estados de resistência à insulina, inclusive o diabetes tipo 2. O receptor de insulina pertence a uma família de receptores de fatores de crescimento que têm atividade tirosina quinase intrínseca. Após a ligação da insulina o receptor sofre autofosforilação em múltiplos resíduos de tirosina. Isto resulta na ativação da quinase do receptor e conseqüente fosforilação em tirosina de um a família de substratos do receptor de insulina (IRS). De forma similar a outros fatores de crescimento, a insulina usa fosforilação e interações proteína-proteína como ferramentas essenciais para transmitir o sinal. Estas interações proteína-proteína são fundamentais para transmitir o sinal do receptor em direção ao efeito celular final, tais como translocação de vesículas contendo transportadores de glicose (GLUT4) do pool intracelular para a membrana plasmática, ativação da síntese de glicogênio e de proteínas, e transcrição de genes específicos.