Elvan Cavac, Luis E. Ramírez-Tapia, and Craig T. Martin, J Biol Chem, 297(3), xxxx-yyyy, 2021. doi: 10.1016/j.jbc.2021.100999 High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation …

High salt transcription of DNA co-tethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA Read more »

Yasaman Gholamalipour, William C. Johnson, & Craig T. Martin, Nucl Acids Res, 47, e118 (7 pages). 2019 In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as …

Efficient inhibition of RNA self-primed extension by addition of competing 3’-capture DNA – improved RNA synthesis by T7 RNA polymerase Read more »

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Yasaman Gholamalipour, Aruni Karunanayake Mudiyanselage, & Craig T. Martin, Nucl Acids Res, 46, 9253-9263, 2018.  Selected by the editors as a “Breakthrough Article.” Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is …

3’ end additions by T7 RNA polymerase are RNA self-templated, distributive, and diverse in character – RNA-Seq analyses Read more »

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Satamita Samanta & Craig T. Martin, J. Biol. Chem. 288, 31993-32003, 2013. It has long been known that during initial transcription of the first 8-10 bases of RNA, complexes are relatively unstable, leading to the release of short abortive RNA transcripts. An …

Insights into the Mechanism of Initial Transcription in E coli RNA Polymerase. Energetic Mechanisms Leading to Initial Complex Instability and Abortive Cycling Read more »

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Luis Ramírez-Tapia & Craig T. Martin, J. Biol. Chem. 287, 37352-37361, 2012. RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially transcribing complexes are characteristically unstable, yielding short abortive products …

New Insights into the Mechanism of Initial Transcription: the T7 RNA Polymerase Mutant P266L Transitions to Elongation at Longer RNA Lengths than Wild Type Read more »

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Ankit V. Vahia & Craig T. Martin, Biochemistry 50, 7015-7022, 2011.  Although the synthesis of RNA from a DNA template is (and must be) a generally very stable process to enable transcription of kilobase transcripts, it has long been known that …

Direct Tests of the Energetic Basis of Abortive Cycling in Transcription Read more »

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Xiaoqing Liu & Craig T. Martin, J. Biol. Chem. 284, 36262-36270, 2009. Transcription machinery from a variety of organisms shows striking mechanistic similarity. Both multi- and single subunit RNA polymerases have evolved an 8-10 base pair RNA:DNA hybrid as a part of …

Transcription Elongation Complex Stability: The Topological Lock Read more »

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Rosemary S. Turingan, Cuihua Liu, Mary E. Hawkins, & Craig T. Martin, Biochemistry 46, 1714-1723, 2007. T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer …

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase Read more »

Rosemary S. Turingan, Karsten Theis, & Craig T. Martin, Biochemistry 46, 6165-6168, 2007.  T7 RNA polymerase undergoes dramatic structural rearrangements in the transition from initiation to elongation. Two models have been proposed for promoter-bound intermediates late in the transition. (i) A subset …

Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase Read more »

Yi Zhou, Deanna M. Navaroli, Metewo Selase Enuameh, & Craig T. Martin, Proc. Natl. Acad.Sci., U.S.A. 104, 10352-10357, 2007. A recent model for the mechanism of intrinsic transcription termination involves dissociation of the RNA from forward translocated (hypertranslocated) states of the complex …

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward translocation mechanism Read more »

Gang Han, Chang-Cheng You, Byoung-jin Kim, Rosemary S. Turingan, Neil S. Forbes, Craig T. Martin, & Vincent M. Rotello, Angew. Chem. Int. Ed. 45, 3165-3169, 2006. See reports in NCI Nanotech News and Nature Materials Light and life: A photolabile gold nanoparticle has been constructed to …

Light-Regulated Release of DNAand Its Delivery to Nuclei by Means of Photolabile Gold Nanoparticles Read more »

Yi Zhou and Craig T. Martin, J. Biol. Chem. 281, 24441-24448, 2006. T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have …

Observed instability of T7 RNA polymerase elongation complexes can be dominated by collision-induced “bumping” Read more »

Gang Han, Nandini S. Chari, Ayush Verma, Rui Hong, Craig T. Martin, and Vincent M. Rotello, Bioconjugate Chem.16 1356-1359, 2005 Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of …

Controlled Recovery of the Transcription of Nanoparticle-Bound DNA by Intracellular Concentrations of Glutathione Read more »

Gang Han, Craig T. Martin, and Vincent M. Rotello, Chem Biol Drug Des.67 78-82, 2005 Positively charged trimethylammonium-modified mixed monolayer protected clusters (MMPCs) interact with DNA by complementary electrostatic binding, serving as efficient DNA delivery systems. The stability of gold nanoparticle-bound DNA …

Stability of Gold Nanoparticle-Bound DNA toward Biological, Physical, and Chemical Agents Read more »

Edward A. Esposito and Craig T. Martin, J. Biol. Chem.279, 44270-44276, 2004 T7 RNA polymerase recognizes a small promoter, binds DNA, and begins the process of transcription by synthesizing short RNA products without releasing promoter contacts. To determine whether the promoter …

Crosslinking of Promoter DNA to T7 RNA Polymerase Does Not Prevent Formation of a Stable Elongation Complex Read more »

Peng Gong, Edward A. Esposito and Craig T. Martin, J. Biol. Chem.279, 44277-44285, 2004 RNA polymerases bind to specific sequences in DNA, melt open duplex DNA around the start site, and start transcription within the initially melted bubble. The initially transcribing …

Initial bubble collapse plays a key role in the transition to elongation in T7 RNA polymerase Read more »

Karsten Theis, Peng Gong and Craig T. Martin, Biochemistry, 43, 12709-12715, 2004 The N-terminal domain of T7 RNA polymerase undergoes large conformational changes in the transition from transcription initiation to elongation. The rigid body displacement of parts of the N-terminal domain (residues …

Topological and conformational analysis of the initiation and elongation complex of T7 RNA polymerase suggests a new twist Read more »

Iaroslav Kuzmine, Philip A. Gottlieb, & Craig T. Martin, J. Biol. Chem.278, 2819-2823, 2003 Unlike DNA polymerases, an RNA polymerase must initiate transcription de novo, that is binding of the initiating (+1) nucleoside triphosphate must be achieved without benefit of the …

Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase Read more »

Craig T. Martin, Andrea Újvári, & Cuihua Liu, Methods Enzymol. 371, 13-33, 2003 This chapter evaluates the fluorescence Spectroscopy technique for mapping melted regions of DNA along the transcription pathway. Fluorescent base analogs provide a relatively non-perturbing probe of very local structural changes within the DNA …

Evaluation of fluorescence spectroscopy methods for mapping melted regions of DNA along the transcription pathway Read more »

Cuihua Liu & Craig T. Martin, J. Biol. Chem.277, 2725-2731, 2002 Footprinting, fluorescence and x-ray structural information from the initial, promoter bound complex of T7 RNA polymerase describes the very beginning of the initiation of transcription, while recent fluorescence and biochemical …

Promoter Clearance by T7 RNA Polymerase: Initial Bubble Collapse and Transcript Dissociation Monitored by Base Analog Fluorescence Read more »

Iaroslav Kuzmine & Craig T. Martin, J. Mol. Biol. 305, 559-566, 2001 In order to begin to understand the mechanism of the initiation of transcription in the model T7 RNA polymerase system, the simplest possible reaction, the synthesis of dinucleotide, has been followed …

Pre-steady State Kinetics of Initiation of Transcription by T7 RNA Polymerase – A New Kinetic Model Read more »

Cuihua Liu & Craig T. Martin, J. Mol. Biol. 308, 465-475, 2001 The various kinetic and thermodynamic models for transcription elongation all require an understanding of the nature of the melted bubble which moves with the RNA polymerase active site. Is the general …

Fluorescence Characterization of the Transcription Bubble in Elongation Complexes of T7 RNA Polymerase Read more »

Iaroslav Kuzmine, Philip A. Gottlieb, & Craig T. Martin, Nucl. Acids Res.29, 2601-2606, 2001 T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago, it was observed that in the presence of …

Structure in Nascent RNA Leads to Termination of Slippage Transcription by T7 RNA Polymerase Read more »

Catherine M. McIntosh, Edward A. Esposito, III, Andrew K. Boal, Joseph M. Simard, Craig T. Martin, & Vincent M. Rotello, J. Am. Chem. Soc.123, 7626-7629, 2001 Efficient recognition of DNA is a prerequisite for the development of biological effectors, including transcription …

Inhibition of DNA transcription using cationic mixed monolayer protected gold clusters Read more »

Manli Jiang, Minqing Rong, Craig Martin, and William T. McAllister, J. Mol. Biol.310, 509-522, 2001 We have explored the effects of a variety of structural and sequence changes in the initiation region of the T7 promoter on promoter function. At promoters …

Interrupting the Template Strand of the T7 Promoter Facilitates Translocation of the DNA During Initiation, Reducing Transcript Slippage and the Release of Abortive Products Read more »

Benjamin F. Weston, Iaroslav Kuzmine, & Craig T. Martin, J. Mol. Biol 272, 21-30, 1997 The determination of various polymerase structures has sparked interest in understanding how the polynucleotide template interacts with the active site. In the primer-independent initiation of transcription, an additional …

Positioning of the Start Site in the Initiation of Transcription by T7 RNA Polymerase Read more »

Andrea Újvári & Craig T. Martin, J. Mol. Biol. 273, 775-781, 1997 The T7 RNA polymerase promoter has been proposed to contain two domains: the binding region upstream of position −5 is recognized through apparently traditional duplex contacts, while the catalytic domain downstream …

Identification of a Minimal Binding Element within the T7 RNA Polymerase Promoter Read more »

Tong Li, Hoi Hung Ho, Maribeth Maslak, Charlie Schick & Craig T. Martin, Biochemistry 35, 3722-3727, 1996 T7 RNA polymerase recognizes a relatively small promoter extending only seventeen base pairs upstream from the start site for transcription. A model for this recognition …

Major Groove Recognition Elements in the Middle of the T7 RNA Polymerase Promoter Read more »

Andrea Újvári & Craig T. Martin, Biochemistry 35, 14574-14582, 1996 Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting (Maslak, M., …

Thermodynamic and Kinetic Measurements of Promoter Binding by T7 RNA Polymerase Read more »

Charlie Schick and Craig T. Martin, Biochemistry 34, 666-672, 1995 The T7, T3, and SP6 RNA polymerases represent a highly homologous family of enzymes that recognize similarly homologous promoter DNA sequences. Despite these similarities, the enzymes are highly specific for their …

Tests of a Model of Specific Contacts in T7 RNA Polymerase-Promoter Interactions Read more »

Maribeth Maslak and Craig T. Martin, Biochemistry, 33, 6918-6924, 1994 The T7 family of DNA-dependent RNA polymerases presents an ideal model system for the study of fundamental aspects of transcription. The small size of the promoter allows a variety of studies …

Effects of Solution Conditions on the Steady State Kinetics of Initiation of Transcription by T7 RNA Polymerase Read more »

Maribeth Maslak, Martha D. Jaworski, and Craig T. Martin, Biochemistry 32, 4270-4274, 1993 The DNA dependent RNA polymerase from bacteriophage T7 is highly specific for a 17 base promoter sequence. Interactions between T7 RNA polymerase and its promoter DNA have been …

Tests of a Model for Promoter Recognition by T7 RNA Polymerase: Thymine Methyl Group Contacts Read more »

Charlie Schick and Craig T. Martin, Biochemistry 32, 4275-4280, 1993 The T7, T3, and SP6 RNA polymerases recognize very similar, yet distinct, promoter sequences. The high homology among the promoter sequences suggests that differential promoter recognition must derive from relatively small …

Identification of Specific Contacts in T3 RNA Polymerase-Promoter Interactions: Kinetic Analysis using Small Synthetic Promoters Read more »

Maribeth Maslak and Craig T. Martin, Biochemistry 32, 4281-4285, 1993 T7 RNA polymerase is highly specific for the initiation of transcription from a relatively small consensus promoter sequence. Previous footprinting studies suggested that the enzyme binds specifically to a fully closed …

Kinetic Analysis of T7 RNA Polymerase Transcription Initiation From Promoters Containing Single Stranded Regions Read more »

Richard V. Prigodich & Craig T. Martin, Biochemistry 29, 8017-8019, 1990 This study demonstrates that the reaction of Fe(II)-EDTA and hydrogen peroxide with the single-stranded nucleic acids d(pT)70 and a 29 base sequence containing a mixture of bases results in substantial …

Reaction of Single-Stranded DNA with Hydroxyl Radical Generated by Iron(II)-Ethylenediaminetetraacetic Acid Read more »

Craig T. Martin and Joseph E. Coleman, Biochemistry 28, 2760-2762, 1989 The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We …

T7 RNA Polymerase Does Not Interact with the 5′-Phosphate of the Initiating Nucleotide Read more »

Daniel K. Muller, Craig T. Martin, and Joseph E. Coleman, Biochemistry 28, 3306-3313, 1989 The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA …

T7 RNA Polymerase Interacts with Its Promoter from One Side of the DNA Helix Read more »