Strong Natural Pausing by RNA Polymerase II within 10 Bases of Transcription Start May Result in Repeated Slippage and Reextension of the Nascent RNA

We find that immediately following transcript initiation, RNA polymerase II pauses at several locations even in the presence of relatively high (200 μM) levels of nucleoside triphosphates. Strong pauses with half-lives of >30 s were observed at +7, +18/19, and about +25 on the template used in these experiments. We show that the strong pause at +7, after the synthesis of 5′-ACUCUCU, leads to repeated cycles of upstream slippage of the RNA-DNA hybrid followed by re-pairing with the DNA and continued RNA synthesis. The resulting transcripts are 2, 4, and 6 bases longer than predicted by the template sequence. Slippage is efficient when transcription is primed with the +1/+2 (ApC) dinucleotide, and it occurs at even higher levels with the +2/+3 primer (CpU). Slippage can occur at high levels with ATP initiation, but priming with CpA (−1/+1) supports very little slippage. This latter result is not simply an effect of transcript length at the point of pausing. Slippage can also occur with a second template on which the polymerase can be paused after synthesizing ACUCU. Slippage is not reduced by an ATP analog that blocks promoter escape, but it is inhibited by substitution of 5Br-U for U in the RNA. Our results reveal an unexpected flexibility of RNA polymerase II ternary complexes during the very early stage of transcription, and they suggest that initiation at different locations within the same promoter gives rise to transcription complexes with different properties

Expression of the Arabidopsis thaliana 2S albumin gene 3 in Saccharomyces cerevisiae

The Arabidopsis thaliana (At) 2S albumin gene 3 (At2S3) has been cloned in YEp13 as a 3.5-kb genomic fragment. To study its expression in Saccharomyces cerevisiae, the accumulation in saturated cultures reached about 0.032% of the yeast total protein, and the product was localized in vacuolar bodies within the cell. The 13-kDa protein was processed to 9- and 4-kDa proteins, as obtained in transgenic tobacco plants

Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1

The requirement of p65/RelA in Hsp70A1A transcription under heat shock.

<p>A) Representative ethidium bromide stained agarose gels showing relative transcripts levels prepared from HeLa cells as indicated determined by RT-PCR. B) Estimation of the effect of p65 knockdown on Hsp70A1A transcription shown in panel A by RT-qPCR using GAPDH as an internal control. One-way ANOVA with Turkey’s post test was used to analyse the data where ***p < 0.001. C) Immunoblot showing downregulation of p65/RelA protein in WCL isolated from HeLa cells pre-treated with sip65 or scramble siRNA. Fold change shown was estimated by densitometric scanning of intensities of bands of p65/RelA vs β-actin. D) Representative ethidium bromide stained agarose gels showing the effects on Hsp70A1A following the treatment of cells as indicated. PMA treated cells were included as positive control. Andro, andrographolide. E) Estimation of intensities of the bands in panel D. GAPDH level was measured as an internal control. One-way ANOVA with Turkey’s post test was used to analyse the data where ***p < 0.001. F) p65/RelA presence in the nucleus following heat shock (plus 45 min recovery at 37°C) or PMA treatment detected by indirect immunofluorescence technique visualised by a confocal microscope. Antibodies against the epitopes were used to detect transiently expressed the FLAG-p65 or his₆-HSF1 protein, respectively. The images were taken in 63x magnification with a 3x optical zoom. HS, heat shock; NHS, non heat shock. AF, alexa flour. The bar graph on the right show the estimates of cells (%) with nuclear p65. Estimation was carried out using student’s <i>t</i> test, *p<0.05. G) Translocation of FLAG-p65 and his₆-HSF1 in isolated cytoplasmic (C) and nuclear (N) protein fractions by immunoblot using anti-FLAG and anti-his₆ antibody, respectively. The α-tubulin protein level was estimated by immunoblot as a cytoplasmic protein marker. Net nuclear translocation of p65/RelA and his₆HSF1 was estimated by subtracting residual cytoplasm contamination in the nuclear fraction equivalent to α-tubulin level.</p

DNA-PKc is required for heat shock mediated transcription induction of Hsp70A1A gene in human cells.

<p>cDNAs prepared from HT1080 cells pretreated with shRNA against catalytic subunit of DNA-PK (shDNA-PKc) were subjected to PCR for testing its effects on the transcription of Hsp70A1A gene using the procedures described in the materials and methods. A) Representative ethidium bromide stained agarose gels showing relative transcript levels of DNA-PKc, Hsp70A1A and β-actin (as loading control). B) Estimation of DNA-PKc and the Hsp70A1A mRNA levels in indicated samples (indicated in panel A) by the RTq-PCR normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyse the data where **p < 0.01. C) Immunoblot showing downregulation of DNA-PKc protein in whole cell lysate isolated from HeLa cells pre-treated with shDNA-PKc or mock treated. Fold change shown was estimated by densitometric scanning of intensities of bands of DNA-PKc vs β-actin. D) Representative agarose gels stained with ethidium bromide showing the effect on Hsp70A1A transcription upon chemical inhibition (100 μM for 24 h) of DNA-PK determined by RT-PCR. E) Estimation of the effect of chemical inhibition DNA-PK by RT-qPCR assay on transcription of Hsp70A1A normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyze the data where **p < 0.001.</p

DNA-PK holoenzyme is required for induction of Hsp70A1A transcription in response to heat shock (HS).

<p>cDNAs prepared from cells pre-treated with Ku70 specific (siKu70), or scrambled siRNA were subjected to PCR to determine expression levels of indicated genes. A) Representative ethidium bromide stained agarose gels indicating the relative levels of Ku70 and Hsp70A1A transcripts. B) The abundance of the indicated transcripts/cDNAs (as indicated in panel A) were estimated by RT-qPCR normalized with normalized with β-actin level. One-way ANOVA with Turkey’s post-test was used to analyse the data where ***p < 0.001. C) Immunoblot showing downregulation of Ku70 protein in whole cell lysate isolated from HeLa cells pre-treated with siKu70 or scramble siRNA. Fold change shown was estimated by densitometric scanning of intensities of bands of Ku70 vs β-actin. D) Representative ethidium bromide stained agarose gels showing the levels of transcripts isolated from cells pre-treated with siKu70, or shDNA-PKc or both siKu70 and shDNA-PKc simultaneously determined by RT-PCR assay. E) Estimation of the intensities of bands shown in panel D through densitometric scanning. The β-actin level was used as the loading control (bottom panel). One-way ANOVA with Turkey’s post-test was used to analyse the data where ***p < 0.001.</p

NFκB modulates Hsp70A1A promoter activity under heat shock.

<p>A) Schematic showing the regions of Hsp70A1A promoter fragments cloned upstream of renila luciferase reporter (Rluc). The locations of heat shock elements (dHSE, distal heat shock element; pHSE, proximal heat shock element), NF-Y and κB consensus sequence (κBc) and a less conserved κB (?) site are indicated with respect to transcription start site (+1, an arrow). The direction of the arrow indicates the direction of transcription. B) RT-PCR assay with transcripts prepared from HEK293 cells 48 h post transfection with the constructs shown in panel ‘A’. Representative ethidium bromide stained agarose gels showing relative levels of indicated transcripts. C) Estimation of the band intensities of luciferase cDNA vs those of GFP and GAPDH as the internal control. D) RT-PCR assay with transcripts prepared from HEK293 cells 48 h posttransfection with the constructs indicated (also shown in panel A, constructs #3 and #3(M)). Representative ethidium bromide stained agarose gels showing relative levels of transcripts as indicated. E) Estimation of the band intensities of lucifearse cDNA vs those of GFP and GAPDH as the internal control. HS, heat shock.</p

Chlorophyll rich biomolecular fraction of A. cadamba loaded into polymeric nanosystem coupled with Photothermal Therapy: A synergistic approach for cancer theranostics.

Development of multifunctional biodegradable nanomaterials to encapsulate hydrophobic drugs and their triggered release in cancer theranostics is a challenge. In the current study, we report the encapsulation of potent anticancer − chlorophyll rich biomolecular fraction from the plant Anthocephalus cadamba into a polymeric nanosystem. The biomolecular fraction was combined with an NIR dye IR-780 to make it photo-thermally active. It was evaluated for its combinatorial (biomolecular extract and photothermal mediated) synergistic cytotoxicity in skin cancer cells. The inherent fluorescence of chlorophyll in the fraction was deployed to understand the cellular uptake and drug release. Cellular uptake of hydrophobic CFAc was enhanced with the aid of nanoformulation. It was observed that photo stability of IR-780 improved when incorporated with CFAc in polymeric nanosystem, which resulted in enhanced photothermal transduction efficiency. The combinational approach exhibited synergistic cytotoxicity which can be applied for skin cancer theranostics

Interaction of p65/RelA with Ku70, DNA-PKc and PARP-1 determined by immunoprecipitation coupled immunoblot experiments.

<p>Whole cell lysates (WCL) of HeLa cells stably expressing FLAG-Ku70 or transiently expressing FLAG-p65/RelA proteins pretreated with heat shock (HS) or not were used. A) Detection of p65/RelA using anti-p65 antibody by immunoblot in immunoprecipitate (IP) obtained from WCL of cells stably expressing FLAG-ku70 using anti-FLAG (M2) antibody. Asterisk denotes a non-specific band. The same membrane was stripped to detect the presence of FLAG-Ku70 protein (lower panel) using anti-FLAG antibody. B) Immunoblot to detect Ku70 or DNA-PKc in immunoprecipitate isolated with anti-FLAG antibody from WCL of cells expressing FLAG-p65/RelA pretreated as indicated. FLAG-p65/RelA level was detected by reblotting the same membrane with the anti-FLAG antibody (FLAG-Ab). The amounts of WCL analyzed as an internal control to identify the target protein band were indicated on the right. C) Immunoblot showing PARP-1 in immunoprecipitate isolated from WCL used in panel B. D) RT-PCR analysis of cDNAs prepared from cells treated with PARP-1 specific siRNA (siPARP-1) or mock to show the effects on Hsp70A1A transcription as indicated by representative agarose gels stained with ethidium bromide. E) The abundance of the indicated transcripts/cDNAs (indicated in panel D) were estimated by RT-qPCR normalized with GAPDH level as internal control. One-way ANOVA with Turkey’s post-test was used to analyse the data where *p < 0.05, **p < 0.01 and ***p < 0.001 respectively. F) Immunoblot showing downregulation of PARP-1 protein in WCL isolated from HeLa cells pre-treated with si-PARP-1 or mock treated. Fold change shown was estimated by densitometric scanning of PARP-1 vs β-Actin bands.</p