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Smita S Patel - One of the best experts on this subject based on the ideXlab platform.
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T7 RNA Polymerase induced bending of promoter dna is coupled to dna opening
Biochemistry, 2006Co-Authors: Guoqing Tang, Smita S PatelAbstract:To initiate transcription, T7 RNA Polymerase (RNAP) forms a specific complex with its promoter DNA and melts several base pairs near the initiation site to form an open complex. Previous gel electrophoresis studies have indicated that the promoter DNA in the initiation complex is bent [Ujvari, A., and Martin, C. T. (2000) J. Mol. Biol. 295, 1173-1184]. Here we use fluorescence resonance energy transfer (FRET) to investigate the conformation of promoter DNA in the closed and open complexes of T7 RNAP. We have used steady state and time-resolved fluorescence approaches to measure the FRET efficiency in a doubly dye-labeled duplex promoter and in a premelted bubble promoter. Changes in the FRET efficiency and hence the DNA end-to-end distance changes are small when the duplex promoter forms a complex with T7 RNAP. On the other hand, FRET changes are relatively larger when the bubble promoter binds T7 RNAP or when initiating nucleotides are added to the duplex promoter-T7 RNAP complex. The shortening of DNA end-to-end distances is indicative of DNA bending in the bubble DNA complex and in the duplex promoter complex with the initiating nucleotides. Our results are consistent with the model in which in the absence of initiating nucleotides there is a distribution of closed and open complexes, and the promoter DNA is bent slightly by <40 degrees in the closed complex but bent more sharply by 86 degrees in the open complex. The energetics of DNA bending suggests that a significant part of the available free energy from promoter and Polymerase interactions is utilized in DNA bending and/or untwisting. We propose that promoter opening occurs spontaneously upon DNA bending and/or untwisting as free energy is gained through interactions of the melted promoter with the T7 RNAP active site.
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rapid binding of T7 RNA Polymerase is followed by simultaneous bending and opening of the promoter dna
Biochemistry, 2006Co-Authors: Guoqing Tang, Smita S PatelAbstract:To form a functional open complex, bacteriophage T7 RNA Polymerase (RNAP) binds to its promoter DNA and induces DNA bending and opening. The objective of this study was to elucidate the temporal coupling in DNA binding, bending, and opening processes that occur during initiation. For this purpose, we conducted a combined measurement of stopped-flow fluorescence anisotropy, fluorescence resonance energy transfer (FRET), and 2-aminopurine fluorescence. Stopped-flow anisotropy measurements provided direct evidence of an intermediate resulting from rapid binding of the promoter to T7 RNA Polymerase. Stopped-flow FRET measurements showed that promoter bending occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly bent. Similarly, stopped-flow 2-aminopurine fluorescence changes showed that promoter opening occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly melted. The indistinguishable observed rate constants of FRET and 2-aminopurine fluorescence changes indicate that DNA bending and opening processes are temporally coupled and these DNA conformational changes take place after the DNA binding step. The results in this paper are consistent with the mechanism in which the initial binding of T7 RNAP to the promoter results in a closed complex, which is then converted into an open complex in which the promoter is both sharply bent and melted.
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peculiar 2 aminopurine fluorescence monitors the dynamics of open complex formation by bacteriophage T7 RNA Polymerase
Journal of Biological Chemistry, 2001Co-Authors: Rajiv P Bandwar, Smita S PatelAbstract:Abstract The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA Polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA Polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at −4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA Polymerase. Based on the recent crystal structure of the T7 RNA Polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at −4 from the guanine at −5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA Polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA Polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, EDc is formed with a K d of 1.8 μm. This complex isomerizes to an open complex, EDo1, in an energetically unfavorable reaction with an equilibrium constant of 0.12. The EDo1 further isomerizes to a more stable open complex, EDo2, with a rate constant around 300 s− 1. Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s− 1.
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inhibition of T7 RNA Polymerase transcription initiation and transition from initiation to elongation are inhibited by T7 lysozyme via a teRNAry complex with RNA Polymerase and promoter dna
Biochemistry, 1997Co-Authors: Amarendra Kumar, Smita S PatelAbstract:The mechanism of transcription repression of T7 RNA Polymerase by T7 lysozyme was investigated using a combination of kinetic and equilibrium methods. HPLC gel-filtration experiments demonstrated complex formation between T7 lysozyme, T7 RNA Polymerase, and promoter DNA. The interactions between the two proteins were quantitated by measuring in real time the changes in protein fluorescence upon binary complex formation using stopped-flow kinetics. Complex formation between T7 lysozyme and the RNA Polymerase was found to occur by a one-step process, with a bimolecular association rate constant of 38 microM-1 S-1 and a dissociation rate constant of 3.5 S-1. These constants provided an equilibrium dissociation constant, Kd, of 92 nM for the Polymerase lysozyme complex. The interactions of the Polymerase with the DNA were studied by stopped-flow kinetics and nitrocellulose equilibrium DNA binding experiments in the absence and in the presence of T7 lysozyme. The results showed that T7 lysozyme did not prevent or change the kinetic or thermodynamic interactions of the RNA Polymerase with the DNA. T7 lysozyme by itself did not bind to the DNA, but since it bound to the RNA Polymerase as well as to the Polymerase DNA complex, transcription repression must involve the formation of the teRNAry complex between T7 lysozyme, T7 RNA Polymerase and the promoter DNA. The effect of T7 lysozyme was most striking on runoff product synthesis which was greatly inhibited whereas the steady-state synthesis of abortive products, limited by Polymerase cycling or RNA dissociation, was relatively unaffected by the presence of T7 lysozyme. Investigation of the pre-steady-state kinetics of transcription in the presence and absence of T7 lysozyme indicated that the inhibition of runoff product synthesis was largely due to inhibition of transcription initiation and transition from initiation to elongation.
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equilibrium and stopped flow kinetic studies of interaction between T7 RNA Polymerase and its promoters measured by protein and 2 aminopurine fluorescence changes
Journal of Biological Chemistry, 1996Co-Authors: Amarendra Kumar, Smita S PatelAbstract:Abstract The mechanism of bacteriophage T7 RNA Polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the Polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the Polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (−4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, where the RNA Polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA Polymerase is specifically and tightly bound to the promoter DNA. Both the Polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA Polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Φ10 and Φ3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k−2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k−2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Φ3.8 a weaker promoter relative to Φ10.
Sue L Povlock - One of the best experts on this subject based on the ideXlab platform.
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vaccinia virus T7 RNA Polymerase expression system for neurotransmitter transporters
Methods in Enzymology, 1998Co-Authors: Sue L PovlockAbstract:Publisher Summary This chapter describes a procedure for the liposome-mediated transfection of the human dopamine transporters (DAT) gene under the control of the T7 promoter into HeLa cells infected with a recombinant vaccinia virus expressing the T7 Polymerase gene. When a strain of vaccinia virus engineered to express the gene for bacteriophage T7 RNA Polymerase is added to cells, it infects the cells and synthesizes the Polymerase in the cytoplasm. The enzyme then acts on T7 promoter-driven target sequences to produce a rapid, high level of expression of a gene inserted downstream of a T7 promoter. Plasmids containing the target DNA are introduced into the cells using Lipofectin, a cationic lipid that spontaneously forms liposomes, which complex with the DNA, fuse with the plasma membrane, and ultimately allow the expression of the desired gene.
David A Scicchitano - One of the best experts on this subject based on the ideXlab platform.
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transcription of dna containing the 5 guanidino 4 nitroimidazole lesion by human RNA Polymerase ii and bacteriophage T7 RNA Polymerase
DNA Repair, 2008Co-Authors: Alexandra Dimitri, Lei Jia, Suse Broyde, David A ScicchitanoAbstract:Damage in transcribed DNA presents a challenge to the cell because it can partially or completely block the progression of an RNA Polymerase, interfering with transcription and compromising gene expression. While blockage of RNA Polymerase progression is thought to trigger the recruitment of transcription-coupled DNA repair (TCR), bypass of the lesion can also occur, either error-prone or error-free. Error-prone transcription is often referred to as transcriptional mutagenesis (TM). Elucidating why some lesions pose blocks to transcription elongation while others do not remains a challenging problem. As part of an effort to understand this, we studied transcription past a 5-guanidino-4-nitroimidazole (NI) lesion, using two structurally different RNA Polymerases, human RNA Polymerase II (hRNAPII) and bacteriophage T7 RNA Polymerase (T7RNAP). The NI damage results from the oxidation of guanine in DNA by peroxynitrite, a well known, biologically important oxidant. It is of structural interest because it is a ring-opened and conformationally flexible guanine lesion. Our results show that NI acts as a partial block to T7RNAP while posing a major block to hRNAPII, which has a more constrained active site than T7RNAP. Lesion bypass by T7RNAP induces base misincorporations and deletions opposite the lesion (C>A>-1 deletion >G >>> U), but hRNAPII exhibits error-free transcription although lesion bypass is a rare event. We employed molecular modeling methods to explain the observed blockage or bypass accompanied by nucleotide incorporation opposite the lesion. The results of the modeling studies indicate that NI's multiple hydrogen-bonding capabilities and torsional flexibility are important determinants of its effect on transcription in both enzymes. These influence the kinetics of lesion bypass and may well play a role in TM and TCR in cells.
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bacteriophage T7 RNA Polymerase transcription elongation is inhibited by site specific stereospecific benzo c phenanthrene diol epoxide dna lesions
Biochemistry, 2001Co-Authors: Richard B Roth, Nicholas E Geacintov, Shantu Amin, David A ScicchitanoAbstract:Benzo[c]phenanthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo[c]phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE−DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA Polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (−)-trans-anti-B[c]PhDE−N6-dA lesion located within the sequence 5‘-CTCTCACTTCC-3‘. The results show that both (−)-trans-anti-B[c]PhDE−N6-dA and (+)-trans-anti-B[c]PhDE−N6-dA block RNA synthesis. Furthermore, both B[c]PhDE−dA stereoisomeric adducts lead to lower levels of initiation of transcription relative to that observed using an unmodified DNA template. In contrast to these results, placement of the adduct on the nontranscribed strand withi...
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site specific benzo a pyrene diol epoxide dna adducts inhibit transcription elongation by bacteriophage T7 RNA Polymerase
Biochemistry, 1994Co-Authors: Deok Joon Choi, Deirdre J Marinoalessandri, Nicholas E Geacintov, David A ScicchitanoAbstract:Benzo[a]pyrene, an extremely potent procarcinogen and mutagen, is metabolized to a variety of products, including the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene. This product of biotransformation reacts with DNA, forming a series of adducts principally at the N2 position of guanine that differ in their stereochemistry and exhibit unique biological properties. In order to gain a better understanding of the effects on RNA synthesis of these adducts, we used purified bacteriophage T7 RNA Polymerase to transcribe a series of templates containing one of four stereoisomerically pure BPDE-guanine lesions--(+)-trans-,(-)-trans-,(+)-cis-anti-N2-BPDE-guanine--or no damaged bases. To construct suitable double-stranded oligodeoxynucleotides for these studies, we annealed an 11-mer containing a site-specific stereoisomerically pure N2-BPDE-guanine adduct, a 37-mer, and a 10-mer to a complementary 58-base sequence of single-stranded DNA. The oligomers were ligated, purified, and reannealed. The resulting DNA template contained the promoter for T7 RNA Polymerase and a BPDE adduct at position +16 following the transcription initiation site. The results of the transcription assays clearly demonstrate that each of the adducts inhibits elongation by T7 RNA Polymerase, but they do so to significantly different extents, depending on the stereochemical characteristics of the BPDE-modified guanine. The order of inhibition is (+)-trans > (-)-trans > (+)-cis > (-)-cis, when the amount of full-length transcript for each is compared to that obtained for an unmodified template. Furthermore, premature termination of RNA synthesis occurs at or near the site of the BPDE lesion as evidenced by the formation of discrete, truncated transcripts. These results might be related to the fact that the pyrenyl moiety of the trans-BPDE adducts is situated in the minor groove of double-stranded DNA, but is quasi-intercalated into the double helix in the case of the cis stereoisomers.(ABSTRACT TRUNCATED AT 250 WORDS)
William T Mcallister - One of the best experts on this subject based on the ideXlab platform.
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effects of substitutions in a conserved dx2gr sequence motif found in many dna dependent nucleotide Polymerases on transcription by T7 RNA Polymerase
Journal of Molecular Biology, 2002Co-Authors: Diane Imburgio, Michael Anikin, William T McallisterAbstract:The region in bacteriophage T7 RNA Polymerase (RNAP) comprising residues 421-425 contains a sequence motif (DX(2)GR) that is conserved among many DNA-dependent nucleotide Polymerases. We have found that alterations in this motif result in enzymes that display weaker retention of the RNA product during transcript initiation, a decreased ability to make the transition to a stable elongation complex, and changes in substrate binding and catalytic activity. Many of these defects are coupled with an altered response to the presence or absence of the non-template strand. The observed constellation of defects supports a role for the motif in interacting with and stabilizing the RNA:DNA hybrid during the early stages of transcript initiation. This is consistent with the position of the motif in a T7 RNAP initiation complex. Although a conserved DX(2)GR sequence motif is also observed in multisubunit RNAPs, the structural organization of the motif and the manner in which it interacts with the RNA:DNA hybrid in the latter enzymes is different from that in T7 RNAP. However, another element in the multisubunit RNAPs that contains a highly conserved arginine residue may play the same role as R425 in T7 RNAP. (c) 2002 Elsevier Science Ltd.
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the specificity loop of T7 RNA Polymerase interacts first with the promoter and then with the elongating transcript suggesting a mechanism for promoter clearance
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Dmitry Temiakov, Pamela E Mentesana, Arkady Mustaev, Sergei Borukhov, William T McallisterAbstract:Abstract During the early stages of transcription, T7 RNA Polymerase forms an unstable initiation complex that synthesizes and releases transcripts 2–8 nt in length before disengaging from the promoter and isomerizing to a stable elongation complex. In this study, we used RNA⋅protein and RNA⋅DNA crosslinking methods to probe the location of newly synthesized RNA in halted elongation complexes. The results indicate that the RNA in an elongation complex remains in an RNA⋅DNA hybrid for about 8 nt from the site of nucleotide addition and emerges to the surface of the enzyme about 12 nt from the addition site. Strikingly, as the transcript leaves its hybrid with the template, the crosslinks it forms with the RNA Polymerase involve a portion of a hairpin loop (the specificity loop) that makes specific contacts with the binding region of the promoter during initiation. This observation suggests that the specificity loop may have a dual role in transcription, binding first to the promoter and subsequently interacting with the RNA product. It seems likely that association of the nascent RNA with the specificity loop facilitates disengagement from the promoter and is an important part of the process that leads to a stable elongation complex.
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characterization of an unusual sequence specific termination signal for T7 RNA Polymerase
Journal of Biological Chemistry, 1998Co-Authors: Biao He, Alexander Kukarin, Dmitry Temiakov, Stephen T Chinbow, Dmitry L Lyakhov, Minqing Rong, Russell K. Durbin, William T McallisterAbstract:Abstract We have characterized an unusual type of termination signal for T7 RNA Polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the non-template strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.
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the stability of abortively cycling T7 RNA Polymerase complexes depends upon template conformation
Biochemistry, 1996Co-Authors: George A Diaz, Minqing Rong, William T Mcallister, Russell K. DurbinAbstract:We have developed a promoter competition assay to determine whether T7 RNA Polymerase dissociates from its template during abortive cycling. We find that the stability of the initiation complex (IC) depends upon the conformation of the promoter, and that the degree to which the template is unwound contributes importantly to the stability of the IC. On linear DNA or a relaxed plasmid template, the stability of the IC is very low (t1/2 30 min) even in the absence of RNA synthesis. These findings are important to our understanding of the transition from the IC to an EC.
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characterization of two types of termination signal for bacteriophage T7 RNA Polymerase
Journal of Molecular Biology, 1994Co-Authors: Lynn E Macdonald, Russell K. Durbin, John J. Dunn, William T McallisterAbstract:Abstract The late bacteriophage T7 terminator (T7-TΦ) encodes an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues; termination occurs at a 3′ G residue just downstream of the U run. In this work, we have explored the features of this signal that are required for efficient termination by T7 RNA Polymerase. Whereas replacement of the template-encoded 3′ G residue with A, C, or U by site-directed mutagenesis had little effect, removal of the U-tract prevented termination. Deletion analysis indicates that the stem-loop and U-tract are not sufficient for termination, and that sequences upstream from the terminator have marked effects on the position and efficiency of termination. A sequence within the human preproparathyroid hormone (PTH) gene that encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure, also serves as an efficient terminator for T7 RNA Polymerase. We have mapped the primary site of termination in the PTH signal to a G residue that lies downstream of the U-rich run (UUUUCUUG). Deletion analysis indicates that the minimal region required for PTH terminator function extends only 23 bp upstream from the termination site, and subcloning of a 31 bp fragment that includes this region of the PTH signal provides efficient termination. A modified form of T7 RNA Polymerase resulting from a single proteolytic cleavage between residues 178 and 179, or mutant Polymerases that are altered in this region of the enzyme, fail to recognize the PTH signal while still terminating at T7-TΦ.
Shmuel Rozenblatt - One of the best experts on this subject based on the ideXlab platform.
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Replication-deficient vaccinia virus encoding bacteriophage T7 RNA Polymerase for transient gene expression in mammalian cells.
Virology, 1995Co-Authors: L S Wyatt, Shmuel RozenblattAbstract:The vaccinia virus/bacteriophage T7 hybrid transient expression system employs a recombinant vaccinia virus that encodes the T7 RNA Polymerase gene, a plasmid vector with a gene of interest regulated by a T7 promoter, and any cell line suitable for infection and transfection. Although high expression in a majority of cells is achieved, the severe cytopathic effects of vaccinia virus and the safety precautions required for use of infectious agents are undesirable features of the system. Here, we report the construction of a highly attenuated and avian host-restricted vaccinia virus recombinant that encodes the T7 RNA Polymerase gene (MVA/T7 pol) and demonstrate the use of the virus for transient expression in mammalian cells. MVA/T7 pol has reduced cytopathic effects compared to the previously used replication-competent vaccinia virus, while providing a high level of gene expression in multiple mammalian cell lines.
