The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori.

Polyhistidine tags (His-tags) are commonly employed in protein purification strategies due to the high affinity and specificity for metal-NTA columns, the relative simplicity of such protocols, and the assumption that His-tags do not affect the native activities of proteins. However, there is a growing body of evidence that such tags can modulate protein structure and function. In this study, we demonstrate that a His-tag impacts DNA complex formation by the C-terminal domain of the α-subunit (αCTD) of Helicobacter pylori RNA polymerase in a metal-dependent fashion. The αCTD was purified with a cleavable His-tag, and complex formation between αCTD, the nickel-responsive metalloregulator HpNikR, and DNA was investigated using electrophoretic mobility shift assays. An interaction between His-tagged αCTD (HisαCTD) and the HpNikR-DNA complex was observed; however, this interaction was not observed upon removal of the His-tag. Further analysis revealed that complex formation between HisαCTD and DNA is non-specific and dependent on the type of metal ions present. Overall, the results indicate that a histidine tag is able to modulate DNA-binding activity and suggests that the impact of metal affinity tags should be considered when analyzing the in vitro biomolecular interactions of metalloproteins.

Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation.

The target of rapamycin complex 1 (TORC1) pathway is a highly conserved signaling pathway across eukaryotes that integrates nutrient and stress signals to regulate the cellular growth rate and the transition into and maintenance of dormancy. The majority of the pathway's components, including the central TOR kinase, have been lost in the apicomplexan lineage, and it is unknown how these organisms detect and respond to nutrient starvation in its absence. Plasmodium falciparum encodes a putative ortholog of the RNA polymerase (Pol) III repressor Maf1, which has been demonstrated to modulate Pol III transcription in a TOR-dependent manner in a number of organisms. Here, we investigate the role of P. falciparum Maf1 (PfMaf1) in regulating RNA Pol III expression under conditions of nutrient starvation and other stresses. Using a transposon insertion mutant with an altered Maf1 expression profile, we demonstrated that proper Maf1 expression is necessary for survival of the dormancy-like state induced by prolonged amino acid starvation and is needed for full recovery from other stresses that slow or stall the parasite cell cycle. This Maf1 mutant is defective in the downregulation of pre-tRNA synthesis under nutrient-limiting conditions, indicating that the function of Maf1 as a stress-responsive regulator of structural RNA transcription is conserved in P. falciparum Recent work has demonstrated that parasites carrying artemisinin-resistant K13 alleles display an enhanced ability to recover from drug-induced growth retardation. We show that one such artemisinin-resistant line displays greater regulation of pre-tRNA expression and higher survival upon prolonged amino acid starvation, suggesting that overlapping, PfMaf1-associated pathways may regulate growth recovery from both artemisinin treatment and amino acid starvation.IMPORTANCE Eukaryote organisms sense changes in their environment and integrate this information through signaling pathways to activate response programs to ensure survival. The TOR pathway is a well-studied signaling pathway found throughout eukaryotes that is known to integrate a variety of signals to regulate organismal growth in response to starvation and other stresses. The human malaria parasite Plasmodium falciparum appears to have lost the TOR pathway over the course of evolution, and it is unclear how the parasite modulates its growth in response to starvation and drug treatment. Here, we show that Maf1, a protein regulated by TOR in other eukaryotes, plays an important role in maintaining the parasite's viability in the face of starvation and other forms of stress. This suggests that PfMaf1 is a component of a yet-to-be-described nutrient and stress response pathway.

RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry.

Circularized oligonucleotides, or coligos, were previously found to serve as RNA polymerase III (Pol III) templates in vitro and in human tissue culture cells. Here we randomized the 12-nucleotide larger loop (L-loop) of a well characterized coligo and found unexpectedly that in vitro transcription by FLAG-Pol III was not significantly affected. This observation allowed us to test the variable of coligo L-loop size separately from the variable of its sequence. Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of randomized sequence, and the smallest loop forced initiation to move into the stem region. To test further the need for any specific sequence we compared seven nucleotide L-loops composed of random, abasic and abasic-acyclic nucleotides, and all supported transcription by Pol III. Transcription of a series of coligos containing twelve contiguous randomized nucleotides placed at different locations within the coligo structure provided further evidence that the stem-loop junction structure is important for precise initiation. Nearly the same transcript pattern was formed in vitro by Pol III from yeast and human cells. Overall, these experiments support structure, rather than L-loop sequence, as the major determinant of coligo transcription initiation by Pol III.

Yeast Bud27 modulates the biogenesis of Rpc128 and Rpc160 subunits and the assembly of RNA polymerase III.

Yeast Bud27, an unconventional prefoldin is reported to affect the expression of nutrient-responsive genes, translation initiation and assembly of the multi-subunit eukaryotic RNA polymerases (pols), at a late step. We found that Bud27 associates with pol III in active as well as repressed states. Pol III transcription and occupancy at the target genes reduce with the deletion of BUD27. It promotes the interaction of pol III with the chromatin remodeler RSC found on most of the pol III targets, and with the heat shock protein Ssa4, which helps in nuclear import of the assembled pol III. Under nutrient-starvation, Ssa4-pol III interaction increases, while pol III remains inside the nucleus. Bud27 but not Ssa4 is required for RSC-pol III interaction, which reduces under nutrient-starvation. In the bud27Δ cells, total protein level of the largest pol III subunit Rpc160 but not of Rpc128, Rpc34 and Rpc53 subunits is reduced. This is accompanied by lower transcription of RPC128 gene and lower RPC160 translation due to reduced association of mRNA with the ribosomes. The resultant alteration in the normal cellular ratio of the two largest subunits of pol III core leads to reduced association of other pol III subunits and hampers the normal assembly of pol III at an early step in the cytoplasm. Our results show that Bud27 is required in multiple activities responsible for pol III biogenesis and activity.

Tamoxifen represses alcohol-induced transcription of RNA polymerase III-dependent genes in breast cancer cells.

Alcohol consumption in women has been associated with an increased risk of breast cancer, particular in estrogen receptor positive (ER+) cases. Deregulation of RNA polymerase III-dependent (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell transformation and tumor formation. Our recent studies demonstrated that alcohol induces Brf1 expression and Pol III gene transcription via ER. Here, we report that Tamoxifen (Tam) inhibits the induction of Brf1 and Pol III genes in ER+ breast cancer cells. Further analysis indicates that alcohol increases c-Jun expression to upregulate the transcription of Brf1 and Pol III genes, whereas Tam reduces c-Jun expression to repress the transcription of Brf1. Repression of cJun decreases cellular levels of ERα and Brf1. Alcohol-dependent increased occupancy of Brf1 in Pol III gene promoters is reduced by Tam. The repression of Brf1 and Pol III genes by Tam reduces alcohol-induced cell proliferation and colony formation. Together, these results indicate that Tam inhibits alcohol-induced Brf1 expression through c-Jun and ERα to downregulate Pol III gene transcription. Our studies uncover a new mechanism of Tam-treated ER+ breast cancer, by which Tam inhibits tumor growth through repressing Pol III gene transcription.

Diagnostic criteria of systemic sclerosis.

Systemic sclerosis (SSc) is a multisystem disease characterized by vascular abnormalities, immune system activation manifested by SSc-specific autoantibodies and disturbances in fibroblast function. The clinical manifestations are highly heterogeneous and commonly include skin thickening, Raynaud's phenomenon, digital ulcers, gastroesophageal reflux disease, interstitial lung disease and cardiac diastolic dysfunction. The diagnosis of SSc in a patient with typical end-organ disease is relatively straight-forward, but is unsatisfactory because it implies that the diagnosis is delayed until irreversible tissue damage is present. Diagnostic criteria are generally designed to facilitate the clinical process and to allow early institution of therapy to relieve symptoms and possibly prevent irreversible damage. Several attempts at defining diagnostic criteria for SSc have been made in the past. Raynaud's phenomenon, SSc-specific autoantibodies and nailfold capillary abnormalities are among the most promising items likely to be retained in a final set of diagnostic criteria. The EULAR Scleroderma Trial and Research group (EUSTAR) is currently in the process of prospectively validating a set of diagnostic criteria for the very early diagnosis of SSc and results are expected in 2015.

Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila.

The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. Here, we show that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. We find that TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, we show that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, we find that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth.

RNA polymerase III under control: repression and de-repression.

The synthesis of tRNA by yeast RNA polymerase III (Pol III) is regulated in response to changing environmental conditions. This control is mediated by Maf1, the global negative regulator of Pol III transcription conserved from yeast to humans. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from recently reported structural and biochemical data on Pol III and Maf1. Efficient Pol III transcription, following the shift of cells from a non-fermentable carbon source to glucose, requires phosphorylation of Maf1. One of the newly identified Maf1 kinases is the chromatin-bound casein kinase II (CK2). Current studies have allowed us to propose an innovative mechanism of Pol III regulation. We suggest that CK2-mediated phosphorylation of Maf1, occurring directly on tDNA chromatin, controls Pol III recycling.

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression.

BACKGROUND: RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter. RESULTS: We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters. CONCLUSION: In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

Protein kinase A regulates RNA polymerase III transcription through the nuclear localization of Maf1.

Maf1 is an essential and specific mediator of transcriptional repression in the RNA polymerase (pol) III system. Maf1-dependent repression occurs in response to a wide range of conditions, suggesting that the protein itself is targeted by the major nutritional and stress-signaling pathways. We show that Maf1 is a substrate for cAMP-dependent PKA in vitro and is differentially phosphorylated on PKA sites in vivo under normal versus repressing conditions. PKA activity negatively regulates Maf1 function because strains with unregulated high PKA activity block repression of pol III transcription in vivo, and strains lacking all PKA activity are hyperrepressible. Nuclear accumulation of Maf1 is required for transcriptional repression and is regulated by two nuclear localization sequences in the protein. An analysis of PKA phosphosite mutants shows that the localization of Maf1 is affected via the N-terminal nuclear localization sequence. In particular, mutations that prevent phosphorylation at PKA consensus sites promote nuclear accumulation of Maf1 without inducing repression. These results indicate that negative regulation of Maf1 by PKA is achieved by inhibiting its nuclear import and suggest that a PKA-independent activation step is required for nuclear Maf1 to function in the repression of pol III transcription. Finally, we report a previously undescribed phenotype for Maf1 in tRNA gene-mediated silencing of nearby RNA pol II transcription.

Distinct mechanisms for repression of RNA polymerase III transcription by the retinoblastoma tumor suppressor protein.

The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAP(C) and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.

Heterologous overexpression and purification of four common subunits of nuclear RNA polymerases I, II and III of Schizosaccharomyces pombe.

Four subunits of Schizosaccharomyces pombe RNA polymerases I-III shared by all three enzymes (Rpb5, Rpb8, Rpb10 and Rpc10 [Rpb12]) have been overexpressed in Escherichia coli expression vectors pQE or pET as hexahistidine fusions. The recombinant proteins have been purified to near homogeneity using metal-chelate affinity chromatography and gel filtration. Homogeneity and identity of the purified protein preparations was demonstrated by denaturing polyacrylamide gel electrophoresis and TOF-MALDI mass spectrometry. The proteins were obtained in large amounts, and their preparations are currently in use for monoclonal antibody production and physico-chemical studies of these individual components of eukaryotic transcription enzymes.

RNA polymerase III transcripts and the PTB protein are essential for the integrity of the perinucleolar compartment.

The perinucleolar compartment (PNC) is a nuclear substructure present in transformed cells. The PNC is defined by high concentrations of certain RNA binding proteins and a subset of small RNAs transcribed by RNA polymerase III (pol III), including the signal recognition particle RNA and an Alu RNA as reported here. To determine if the PNC is dependent on pol III transcription, HeLa cells were microinjected with the selective pol III inhibitor, Tagetin. This resulted in disassembly of the PNC, whereas inhibition of pol I by cycloheximide or pol II by alpha-amanitin did not significantly affect the PNC. However, overexpression of one of the PNC-associated RNAs from a pol II promoter followed by injection of Tagetin blocked the Tagetin-induced PNC disassembly, demonstrating that it is the RNA rather than pol III activity that is important for the PNC integrity. To elucidate the role of the PNC-associated protein PTB, its synthesis was inhibited by siRNA. This resulted in a reduction of the number of PNC-containing cells and the PNC size. Together, these findings suggest, as a working model, that PNCs may be involved in the metabolism of specific pol III transcripts in the transformed state and that PTB is one of the key elements mediating this process.

[Study on expression of cell cycle-related genes in subclonal cell lines of human cervical carcinoma].

OBJECTIVE: To explore the role of expression of cell cycle-related genes in cervical carcinoma cell lines. METHODS: A series of expression microarray analysis of two homologous cervical carcinoma subclonal cell lines were initiated by cDNA microarray which represent a set of 234 human cell cycle-related genes. RESULTS: In normal medium, the percent of G(1) phase in CS03 cells was higher than in CS07 cells dramatically, but the percent of S phase in CS07 cells was more than in CS03 cells. After cultured 48 h in serum-free medium, the percent of apoptosis cells (sub-G(1) phase) in CS03 cells was higher than in CS07 cells significantly and increased with time. By applying this cDNA microarray, we identified 3 differentially expressed genes in two homologous cell lines, which were BN51, hsp90 and Mcl-1 genes, further identified 3 upregulated genes in CS07 cell line, the ratio of Cy5/Cy3 was 0.480, 0.479 and 0.490 respectively. CONCLUSION: Differential expression of BN51, hsp90 and Mcl-1 genes is associated to various growth patterns of human cervical carcinoma cells.

RNA polymerase III transcription factor TFIIIC2 is overexpressed in ovarian tumors.

Most transformed cells display abnormally high levels of RNA polymerase (pol) III transcripts. Although the full significance of this is unclear, it may be fundamental because healthy cells use two key tumor suppressors to restrain pol III activity. We present the first evidence that a pol III transcription factor is overexpressed in tumors. This factor, TFIIIC2, is a histone acetyltransferase that is required for synthesis of most pol III products, including tRNA and 5S rRNA. TFIIIC2 is a complex of five polypeptides, and mRNAs encoding each of these subunits are overexpressed in human ovarian carcinomas; this may explain the elevated TFIIIC2 activity that is found consistently in the tumors. Deregulation in these cancers is unlikely to be a secondary response to rapid proliferation, because there is little or no change in TFIIIC2 mRNA levels when actively cycling cells are compared with growth-arrested cells in culture. Using purified factors, we show that raising the level of TFIIIC2 is sufficient to stimulate pol III transcription in ovarian cell extracts. The data suggest that overexpression of TFIIIC2 contributes to the abnormal abundance of pol III transcripts in ovarian tumors.

A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts.

The first protein that binds to all newly synthesized RNA polymerase III transcripts is a highly conserved phosphoprotein known as the La autoantigen. Although binding by the yeast La protein Lhp1p to pre-tRNAs is required for the normal pathway of tRNA maturation, the role of the La protein in the biogenesis of other polymerase III transcripts has been unclear. We identified a mutation in a novel component of the U6 snRNP that causes yeast cells to require Lhp1p for growth. This protein, Lsm8p, is a member of a family of proteins, known as Sm-like proteins, that shares two conserved motifs with the core Sm proteins of the U1, U2, U4 and U5 snRNPs. The lsm8-1 cells have drastically reduced levels of the mature U6 snRNP, consistent with a defect in U6 snRNP assembly. In these cells, Lhp1p stabilizes newly synthesized U6 RNA, thus facilitating assembly of the RNA into the U6 snRNP. These results provide evidence that Lhp1p is a molecular chaperone for polymerase III-transcribed RNAs and implicate Lsm8p as a key component in the very early steps of U6 snRNP assembly.

Expression and purification of recombinant hRPABC25, hRPABC17, and hRPABC14.4, three essential subunits of human RNA polymerases I, II, and III.

Transcription of eukaryotic genes is performed by RNA polymerases I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. Eukaryotic RNA polymerases are large macromolecular complexes composed of multiple subunits. Among these subunits, five are shared by all RNA polymerases and are essential for cell growth and viability. Remarkably, the human common subunits are structurally conserved and functionally interchangeable with their yeast homologues and are believed to play an important role in the assembly of the three transcription complexes. To understand the structure and function of human RNA polymerases, we overexpressed the common subunits hRPABC25, hRPABC17, and hRPABC14.4 as hexahistidine fusions in Escherichia coli. The recombinant proteins were purified using metal-chelate affinity chromatography on Ni-NTA resin and gel filtration. Depending on the subunit, the yield was 5-17 mg of purified recombinant protein per liter of culture medium. The purified proteins were of high quality and sufficient quantity for structural studies, as demonstrated by the successful crystallization of hRPABC17 and hRPABC14.4. The expression and purification of the common subunits hRPABC25, hRPABC17, and hRPABC14. 4 will make possible their structural analysis with X-ray crystallography and nuclear magnetic resonance, providing important insights into the structure and function of the three human RNA polymerases.

p53 is a general repressor of RNA polymerase III transcription.

p53 is a major tumour suppressor that is inactivated in a large proportion of human cancers. We show that p53 serves as a general repressor of transcription by RNA polymerase (pol) III. It can inhibit the synthesis of a range of essential small cellular RNAs including tRNA, 5S rRNA and U6 snRNA, as well as viral products such as the adenovirus VAI RNA. Fibroblasts derived from p53 knock-out mice display a substantial increase in pol III transcriptional activity. Endogenous cellular p53 is shown to interact with the TATA-binding protein (TBP)-containing general factor TFIIIB, thereby compromising its function severely. However, assembly of TFIIIB into a pre-initiation complex confers substantial protection against the inhibitory effects of p53. Since TFIIIB is an essential determinant of the biosynthetic capacity of cells, its release from repression by p53 may contribute to a loss of growth control during the development of many tumours.

Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth.

The retinoblastoma family of proteins, also known as pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130. Pocket proteins control growth processes in many cell types, and this has been linked to the ability of pocket proteins to interact with a multitude of cellular proteins that regulate gene expression at various levels. By regulating gene expression, pocket proteins control cell cycle progression, cell cycle entry and exit, cell differentiation and apoptosis. This review will focus on the mechanisms of regulation of pocket proteins and how modulation of pocket protein levels and phosphorylation status regulate association with their cellular targets. The coordinated regulation of pocket proteins provides the cells with a competence mechanism for passage through certain cell growth and differentiation transitions.

Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression.

We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex.