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"New Insights into the Mechanism of Initial Transcription: the T7 RNA
Polymerase Mutant P266L Transitions to Elongation at Longer RNA Lengths than
Wild Type," 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 on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e., at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multi-subunit RNA polymerases are discussed.
"Direct Tests of the Energetic Basis of Abortive Cycling in Transcription," 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 during initial transcription of the first 8-10 bases of RNA complexes are relatively unstable, leading to the release of short abortive RNA transcripts. A wealth of structural data in the past decade has led to specific mechanistic models elaborating an earlier "stressed intermediate" model for initial transcription. In this study, we test fundamental predictions of each of these models in the simple model enzyme T7 RNA polymerase. Nicking or gapping the nontranscribed template DNA immediately upstream of the growing hybrid yields no systematic reduction in abortive falloff, demonstrating clearly that compaction or "scrunching" of this DNA is not a source of functional instability. Similarly, transcription on DNA in which the nontemplate strand in the initially transcribed region is either mismatched or removed altogether leads to at most modest reductions in abortive falloff, indicating that expansion or "scrunching" of the bubble is not the primary driving force for abortive cycling. Finally, energetic stress derived from the observed steric clash of the growing hybrid against the N-terminal domain contributes at most mildly to abortive cycling, as the addition of steric bulk (additional RNA bases) at the upstream end of the hybrid does not lead to predicted positional shifts in observed abortive patterns. We conclude that while structural changes (scrunching) clearly occur in initial transcription, stress from these changes is not the primary force driving abortive cycling.
"Transcription Elongation Complex Stability: The Topological Lock," 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 a stably transcribing elongation complex. Through characterization of halted complexes that can readily carry out homopolymeric slippage synthesis, this study reveals that T7 RNA polymerase elongation complexes containing only a 4 base pair hybrid can nevertheless be more stable than those with the normal 8 base pair hybrid. We propose that a key feature of this stability is the topological threading of RNA through the complex and/or around the DNA template strand. The data are consistent with forward translocation as a mechanism to allow unthreading of the topological lock, as can occur during programmed termination of transcription.
"Transcription: Initiation," Craig T. Martin & Olga S. Mironova, Wiley Encyclopedia of Chemical Biology, Vol 4 (Editor: Tadhg P. Begley), 584-595, 2009.
Initiation of transcription is a complex process, requiring melting of the DNA duplex at a sequence-defined location, de novo synthesis of an initial dinucleotide, polymerization/growth from that dinucleotide to a larger oligonucleotide in an extended RNA-DNA hybrid, displacement and threading of the 5' end of the RNA into an exit channel, and release of the initial promoter contacts. These events are necessarily driven energetically by growth of the RNA-DNA hybrid. It has long been expected and is now becoming structurally clear that stress inherent in this transformation leads to instability during this phase and is a source of the abortive release of short RNA products that is characteristic of RNA polymerases. That apparently unrelated single and multisubunit RNA polymerases share extensive mechanistic (but not structural) homology, likely reflects the demands of the process.
"Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward translocation mechanism," 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 [Yarnell & Roberts (1999) Science, 284, 611-615]. The current study demonstrates that halted elongation complexes of T7 RNA polymerase in the absence of termination signals can also dissociate via a forward translocation mechanism. Shortening of the downstream DNA, or the introduction of a stretch of mismatched DNA immediately downstream of the halt site, reduces a barrier to forward translocation and correspondingly reduces the lifetime of halted complexes. Conversely, introduction of a crosslink downstream of the halt site increases the same barrier and leads to an increase in complex lifetime. Introduction of a mismatch within the bubble reduces a driving force for forward translocation and correspondingly increases the lifetime of the complex, but only for mismatches at the upstream edge of the bubble, as predicted by the model. Mismatching only the 2 most upstream of the 8 bases in the bubble provides a maximal increase in complex stability, suggesting that dissociation occurs primarily from early forward translocated states. Finally, addition in trans of an oligonucleotide complementary to the nascent RNA just beyond the hybrid complements the loss of driving force derived from placement of a mismatch within the bubble, confirming the expected additivity of effects. Thus, forward translocation is likely a general mechanism for dissociation of elongation complexes, both in the presence and absence of intrinsic termination signals.
"Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase"," 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 of promoter interactions are maintained through completion of the protein conformational (twist) change, and (ii) concerted movement (shift) of all promoter-binding elements away from the growing DNA-RNA hybrid leads to an open intermediate, with large-scale domain rotations deferred until after promoter release. Fluorescence resonance energy transfer measurements provide very strong support for the latter.
"Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase"," 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 distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.
"Observed instability of T7 RNA polymerase elongation complexes can be dominated by collision-induced "bumping"," 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 shown lifetimes that are much shorter than those of the multi-subunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 to 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter, can a second polymerase bind, initiate, and displace the leading complex.
"Mechanism of instability in abortive cycling by T7 RNA polymerase," Peng Gong and Craig T. Martin, J. Biol. Chem. 281, 23533-23544, 2006.
Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions 14 and -13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.
"Light-Regulated Release of DNAand Its Delivery to Nuclei by Means of Photolabile Gold Nanoparticles,"
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
(supplementary info available).
See reports in NCI Nanotech News and Nature Materials
"Stability of Gold Nanoparticle-Bound DNA toward Biological, Physical, and Chemical Agents,"
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 toward biological, physical, and chemical agents is investigated. The MMPC-bound DNA is efficiently protected from DNAse I digestion and experiences nicking/cleavage-induced morphology changes with higher concentrations of DNAse I. Significant protection of MMPC-bound DNA was also observed in a physical sonication assay. However, the MMPC-bound DNA was found to show enhanced cleavage upon exposure to chemically induced radicals. The latter may indicate that bound DNA is bent and wrapped on the surface of the cationic MMPC.
"Controlled Recovery of the Transcription of Nanoparticle-Bound DNA by Intracellular Concentrations of Glutathione,"
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 T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.
"Structure and Function in Promoter Escape by T7 RNA Polymerase,"
Craig T. Martin, Edward A. Esposito, Karsten Theis, & Peng Gong, Prog Nucl Acids Res & Mol Biol, Kivie Moldave, Ed., 80 323-347, 2005
"Topological and conformational analysis of the initiation and elongation complex of T7 RNA polymerase suggests a new twist,"
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 72-152 and 204-258) has been described as a screw motion composed of a rotation by 140° and a translation of >20 Ā along the rotation axis. Protein-protein interactions between residues 23-42 and the C-terminal domain are present in both the initiation and the elongation complex. Assuming that these interactions are retained during the transition between the two states, we find that topological constraints require a right-handed 220° screw motion of the N-terminal rigid body rather than the proposed 140° left-handed screw motion. In the initiation complex, a loop (residues 153-203) extruding from the N-terminal rigid domain wraps around the N-terminal 30 residues. Assuming the Nterminal rigid domain stays folded during the transition, the N-terminus has to pass through this loop before the rigid domain can undergo the translation leading to the elongation complex. Based on these topological constraints, we suggest an alternate sequence of conformational changes leading from transcription initiation to elongation in T7 polymerase.
"Initial bubble collapse plays a key role in the transition to elongation in T7 RNA polymerase," 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 complex is relatively unstable, releasing short abortive products. After synthesis of a minimal length of RNA (about 10-12 bases in the T7 system), RNA polymerases complete the transition to a processive (highly stable) elongation phase and lose the initial promoter contacts. The current study strongly supports a model for T7 RNA polymerase in which initial bubble collapse from position ?4 to position +3 is responsible for initiating RNA displacement in the transition process. More specifically, collapse of the bubble from position ?4 to position ?1 indirectly and energetically facilitates the direct strand invasion offered by collapse at positions +1 to +3. Parallel work shows that promoter release, another key event occurring during this stage of transcription, begins after translocation to position +8 and is largely complete by translocation to about position +12. The timing of promoter release agrees with the timing of initial bubble collapse determined by our previous fluorescence studies, suggesting that these two events are closely related.
"Crosslinking of Promoter DNA to T7 RNA Polymerase Does Not Prevent Formation of a Stable Elongation Complex," Edward A. Esposito and Craig T. Martin, J. Biol. Chem. 279, 44270-44276, 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 complex is relatively unstable, releasing short abortive products. After synthesis of a minimal length of RNA (about 10-12 bases in the T7 system), RNA polymerases complete the transition to a processive (highly stable) elongation phase and lose the initial promoter contacts. The current study strongly supports a model for T7 RNA polymerase in which initial bubble collapse from position -4 to position +3 is responsible for initiating RNA displacement in the transition process. More specifically, collapse of the bubble from position -4 to position =1 indirectly and energetically facilitates the direct strand invasion offered by collapse at positions +1 to +3. Parallel work shows that promoter release, another key event occurring during this stage of transcription, begins after translocation to position +8 and is largely complete by translocation to about position +12. The timing of promoter release agrees with the timing of initial bubble collapse determined by our previous fluorescence studies, suggesting that these two events are closely related.
"Evaluation of fluorescence spectroscopy methods for mapping melted regions of DNA along the transcription pathway," Craig T. Martin, Andrea Újvári, & Cuihua Liu, Methods Enzymol. 371, 13-33, 2003.
"Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase," 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 cooperative binding energetics of an associated primer. Since a single Watson-Crick base pair is not stable in solution, RNA polymerases might be expected to provide additional stabilizing interactions to facilitate binding and positioning of the initiating (priming) nucleoside triphosphate at position +1. Consistent with base-specific stabilizing interactions, of the seventeen T7 RNA polymerase promoters in the phage genome, fifteen begin with guanine. In this work, we demonstrate that the purine N7 is important in the utilization of the initial substrate GTP. The fact that on a template encoding AG as the first two bases in the transcript (as in the remaining two of the T7 genome), transcription starts predominantly (but not exclusively) at the G at position +2 additionally implicates the purine O6 as an important recognition element in the major groove. Finally, results suggest that these interactions serve primarily to position the initiating base in the active site. It is proposed that T7 RNA polymerase interacts directly with the Hoogsteen side of the initial priming GTP (most likely via an interaction with an arginine side chain in the protein) to provide the extra stability required at this unique step in transcription.
"Promoter Clearance by T7 RNA Polymerase: Initial Bubble Collapse and Transcript Dissociation Monitored by Base Analog Fluorescence," Cuihua Liu & Craig T. Martin, J. Biol. Chem. 277, 2725-2731, 2002.
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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 studies paint a preliminary picture of an elongation complex. The current work focuses on the transition from an initially transcribing, promoter bound complex, to an elongation complex clear of the promoter. Fluorescence quenching is used to follow the melted state of the DNA bubble, and a novel approach employing a locally mismatched fluorescent base analog reports on the local structure of the heteroduplex. Fluorescent base analogs placed at positions ?2 and ?1 of the promoter indicate that this initially melted, nontranscribed region remains melted as the polymerase translocates through to position +8. In progressing to position +9, this region of the DNA bubble begins to collapse. Probes placed at positions +1 and +2 of the template strand indicate that the 5' end of the RNA remains in a heteroduplex as the complex translocates to position +10. Subsequent translocation leads to sequential dissociation of the first two bases of the RNA. These results show that the initially transcribing complex bubble can reach a size of up to 13 base pairs, and a maximal heteroduplex length of 10 base pairs. They further indicate that initial bubble collapse precedes dissociation of the 5' end of the RNA.
"Interrupting the Template Strand of the T7 Promoter Facilitates Translocation of the DNA During Initiation, Reducing Transcript Slippage and the Release of Abortive Products," 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 in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region, and if there is a C residue at that position initiation commences at that site with GTP. This effect is observed regardless of the surrounding sequence and whether or not the consensus sequence is present in the non-template (NT) strand. Although the sequence of the DNA surrounding the start site is not critical for start site selection it is important for melting of the promoter and stabilization of the initiation complex. At consensus promoters in which the integrity of T strand is interrupted by nicks or gaps between ?5 and ?2, the enzyme is able to insert the 3' end of the interrupted T strand into the active site and to localize the start site at +1 correctly. However, on gapped promoters in which the T strand base at ?1 has been removed initiation occurs not at the C at +1 (which would now be at the 3' terminus of the T strand) but at the next C, consistent with a need for a base just upstream in the T strand to position the start site. Strikingly, the synthesis of poly(G) products (which arise by transcript slippage) is dramatically reduced or eliminated on nicked or gapped promoters. Furthermore, interrupting the T strand results in a defect in displacement of the nascent RNA and a decrease in the release of abortive initiation products 8-11 nt in length. These effects are attributed to a change in the manner in which the nicked T strand is retained in the initiation complex and a lowered barrier to translocation of the template.
"Inhibition of DNA transcription using cationic mixed monolayer protected gold clusters," 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 and translation regulators, transfection vectors, and DNA sensors. To provide an effective scaffold for multivalent interactions with DNA, we have fabricated mixed monolayer protected gold clusters (MMPCs) functionalized with tetraalkylammonium ligands that can interact with the DNA backbone via charge complementarity. Binding studies indicate that the MMPCs and DNA form a charge neutralized, nonaggregated assembly. The interactions controlling these assemblies are highly efficient, completely inhibiting transcription by T7 RNA polymerase in vitro.
"Structure in Nascent RNA Leads to Termination of Slippage Transcription by T7 RNA Polymerase," 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 only GTP as a substrate, on a template encoding the initial sequence GGGA?, T7 RNA polymerase will synthesize a "ladder" of poly?G RNA products (Martin, C. T., Muller, D. K. & Coleman, J. E. (1988). Biochemistry 27, 3966-3974.). At each step, the ratio of elongation to product release is consistently about 0.75 until the RNA reaches a length of about 13?14 nucleotides, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore become sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7?deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.
"Fluorescence Characterization of the Transcription Bubble in Elongation Complexes of T7 RNA Polymerase
," Cuihua Liu & Craig T. Martin, J. Mol. Biol. 308, 465-475, 2001.
Reviewed in Current Opinions in Structural Biology
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 nature of the bubble system-dependent or are there common energetic requirements which constrain a bubble in any RNA polymerases? T7 RNA polymerase is one of the simplest RNA polymerases and is the system for which we have the most high resolution structural information. However, there is no high resolution information available for a stable elongation complex. In order to directly map melted regions of the DNA in a functionally paused elongation complex, we have introduced fluorescent probes site-specifically into the DNA. Like 2?aminopurine, which substitutes for adenine, the fluorescence intensity of the new probe, pyrrolo?dC, which substitutes for cytosine, is sensitive to its environment. Specifically, the fluorescence is quenched in duplex DNA relative to its fluorescence in single stranded DNA, such that the probe provides direct information on local melting of the DNA. Placement of this new probe at specific positions in the nontemplate strand shows clearly that the elongation bubble extends about eight bases upstream of the pause site, while 2?aminopurine probes show that the elongation bubble extends only about one nucleotide downstream of the last base incorporated. The positioning of the active site very close to the downstream edge of the bubble is consistent with previous studies and with similar studies of the promoter bound, pre-initiation complex. The results show clearly that the RNA:DNA hybrid can be no more than eight nucleotides in length, and characterization of different paused species suggests preliminarily that these dimensions are not sequence or position dependent. Finally, the results confirm that the ternary complex is not stable with short lengths of transcript, but persists for a substantial time when paused in the middle or at the (runoff) end of duplex DNA.
& "Pre-steady State Kinetics of Initiation of Transcription by T7 RNA Polymerase - A New Kinetic Model
," 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 by quench-flow kinetics, and numerical integration of mechanism-specific rate equations has been used to test specific kinetic models. In order to fit the observed time dependence in the pre-steady state kinetics, a model for dinucleotide synthesis is proposed in which rebinding of the dinucleotide to the enzyme-DNA complex must be included. Separate reactions using dinucleotide as a substrate confirm this mechanism and the determined rate constants. The dinucleotide rebinding observed as inhibition under these conditions forms a productive intermediate in the synthesis of longer transcripts, and must be included in future kinetic mechanisms. The rate limiting step leading to product formation shows a substrate dependence consistent with the binding of two substrate GTP molecules, and at saturating levels of GTP, is comparable in magnitude to the product release rate. The rate of product release shows a positive correlation with the concentration of GTP, suggesting that the reaction shows base-specific substrate activation. The binding of another substrate molecule, presumably via interaction with the triphosphate binding site, likely facilitates displacement of the dinucleotide product from the complex.
A printer error at the journal omitted two equations in the PDF version of the manuscript. Download page with the correct equations here.
"Evidence for DNA bending at the T7 RNA polymerase promoter," Andrea Újvári & Craig T. Martin, J. Mol. Biol. 295, 1173-84, 2000.
T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. In the current study, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A complementary set of experiments employing differential phasing from an array of phased A?tracts provides further evidence for both intrinsic and polymerase-induced bends in the T7 RNA polymerase promoter DNA. The bend in the complex is predicted to be about 40-60° and to be centered around positions ?2 to +1, at the start site for transcription, while the intrinsic bend is much smaller (about 10°). These results, viewed in the light of a recent crystal structure for the complex, suggest a mechanism by which binding leads directly to bending. Bending at the start site would then facilitate the melting necessary to initiate transcription.
"Identification of a Minimal Binding Element within the T7 RNA Polymerase Promoter," 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 of position -5 is bound in a melted configuration. This model is tested by following polymerase binding to a series of synthetic oligonucleotides representing truncations of the consensus promoter sequence. The increase in the fluorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations, and allows the determination of both increases and decreases in the dissociation constant. The best fit value of Kd = 4.0 nM for the native promoter is in good agreement with previous fluorescence and steady state measurements. Deletion of the downstream DNA up to position -1 or to position -5 leads to a five-fold increase in binding, while further sequential single-base deletions upstream result in 20 and 500-fold decreases in binding. These results indicate that the (duplex) region of the promoter upstream of and including position -5 is both necessary and sufficient for tight binding, and represents the core binding element of the promoter. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. We also show that the presence of magnesium is necessary for optimal binding, but not for specific enzyme-promoter complex formation, and we propose that magnesium is not required for melting of the promoter.
"Positioning of the Start Site in the Initiation of Transcription by T7 RNA Polymerase," 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 question arises as to how the complex directs the first two bases of the template uniquely into the active site. Recent studies in the model RNA polymerase from bacteriophage T7 demonstrate that upstream duplex contacts provide at least some of the binding specificity and suggest that the enzyme interacts with the template strand in a melted context near the start site for transcription. The current work probes the role of the template strand in positioning of the first two templating bases during initiation. The results suggest that such positioning is not rate limiting in steady state turnover, and that the insertion of a very large and flexible linker three or four bases upstream of the start site has no significant effect on the fidelity of start site selection. The insertion of linkers immediately adjacent to the start site, however, does significantly decrease the fidelity of start site selection (as evidenced by a large increase in misinitiation at position +2, with little change in the observed rate of correct initiation), suggesting that some of the nontranscribed template DNA does help to position the first two templating bases into the active site of the RNA polymerase. Finally, incorporation of an abasic site at position -1 yields a similar decrease in initiation fidelity, suggesting a role for stacking of the bases at positions -1 and +1.
"Thermodynamic and Kinetic Measurements of Promoter Binding by T7 RNA Polymerase," Andrea Újvári & Craig T. Martin, Biochemistry 35, 14574-14582, 1996.
"Major Groove Recognition Elements in the Middle of the T7 RNA Polymerase Promoter," Tong Li, Hoi Hung Ho, Maribeth Maslak, Charlie Schick & Craig T. Martin, Biochemistry 35, 3722-3727, 1996.
"Identification of Essential Amino Acids Within the Proposed
CuA Binding Site in Subunit II of Cytochrome c
Oxidase," Henry Speno, M. Reza Taheri, Derek Sieburth, and Craig T.
Martin, J. Biol. Chem. 270, 25363-25369, 1995.
"Tests of a Model of Specific Contacts in T7 RNA Polymerase-Promoter Interactions," Charlie Schick and Craig T.
Martin, Biochemistry 34, 666-672, 1995.
"Effects of Solution Conditions on the Steady State Kinetics of Initiation of Transcription by T7 RNA Polymerase," Maribeth Maslak and Craig T. Martin, Biochemistry, 33, 6918-6924, 1994.
"Kinetic Analysis of T7 RNA Polymerase Transcription Initiation From Promoters Containing Single Stranded Regions,"
Maribeth Maslak and Craig T. Martin, Biochemistry 32, 4281-4285, 1993.
"Identification of Specific Contacts in T3 RNA
Polymerase-Promoter Interactions: Kinetic Analysis using Small
Synthetic Promoters," Charlie Schick and Craig T. Martin,
Biochemistry 32, 4275-4280, 1993.
"Tests of a Model for Promoter Recognition by T7 RNA Polymerase: Thymine Methyl Group Contacts," Maribeth Maslak, Martha D. Jaworski, and Craig T. Martin, Biochemistry 32, 4270-4274, 1993.
"Reaction of Single-Stranded DNA with Hydroxyl Radical Generated by Iron(II)-Ethylenediaminetetraacetic Acid," Richard V. Prigodich & Craig T. Martin, Biochemistry 29, 8017-8019, 1990.
"T7 RNA Polymerase Interacts with Its Promoter from One Side of the DNA Helix," Daniel K. Muller, Craig T. Martin, and Joseph E. Coleman, Biochemistry 28, 3306-3313, 1989.
"T7 RNA Polymerase Does Not Interact with the 5'-Phosphate of the Initiating Nucleotide," Craig T. Martin and Joseph E. Coleman, Biochemistry 28, 2760-2762, 1989.
"Processivity of Proteolytically Modified Forms of T7 RNA Polymerase," Daniel K. Muller, Craig T. Martin, and Joseph E.
Coleman, Biochemistry 27, 5763-5771, 1988.
"Processivity in Early Stages of Transcription by T7 RNA Polymerase," Craig T. Martin, Daniel K. Muller, and Joseph E. Coleman, Biochemistry 27, 3966-3974, 1988.
"On the Nature of Cysteine Coordination to CuA in Cytochrome c Oxidase," Craig T. Martin, Charles P. Scholes,
and Sunney I. Chan, J. Biol. Chem. 263, 8420-8429, 1988.
"A Proposal for the Site and Mechanism of Redox-Linked Proton
Translocation in Cytochrome c Oxidase," Sunney I. Chan, Peter
Mark Li, Thomas Nilsson, Jeff Gelles, David F. Blair, and Craig T.
Martin, Prog. Clin. Biol. Res. 274, 731-747, 1988.
"Kinetic Analysis of T7 RNA Polymerase-Promoter Interactions with Small Synthetic Promoters," Craig T. Martin and Joseph E. Coleman, Biochemistry 26, 2690-2696, 1987.
"Zinc Metalloproteins Involved in Replication and
Transcription," David P. Giedroc, Kathleen M. Keating, Craig T.
Martin, Kenneth R. Williams, and Joseph E. Coleman, J. Inorg.
Biochem. 28, 155-169, 1986.
"Transcription by T7 RNA Polymerase Is Not Zinc-Dependent and Is Abolished on Amidomethylation of Cysteine-347", Garry C. King, Craig T. Martin, Thang T. Pham, and Joseph E. Coleman, Biochemistry 25, 36-40, 1986.
"The Identification of Histidine Ligand(s) to Cytochrome a in Cytochrome c Oxidase," Craig T. Martin, Charles P.
Scholes, and Sunney I. Chan, J. Biol. Chem. 260, 2857-2861, 1985.
"The Metal Centers of Cytochrome c Oxidase: Structure
and Function," Sunney I. Chan, Craig T. Martin, Hsin Wang, David F.
Blair, Jeff Gelles, Gary W. Brudvig, and Tom H. Stevens, in
Biochemical and Biophysical Studies of Proteins and Nucleic
Acids, Tung-Bin Lo, Teh-Yung Liu, and Choh-Hao Li, eds.,
Elsevier, New York, 219-239, 1984.
"Energetics and Molecular Dynamics of the Proton Pumping
Photocycle in Bacteriorhodopsin," Robert R. Birge, Albert F.
Lawrence, Thomas M. Cooper, Craig T. Martin, David F. Blair, and
Sunney I. Chan, in Nonlinear Electrodynamics in Biological Systems,
W. Ross Adey and Albert F. Lawrence, eds., Plenum, New York, 107-120,
"Structural Studies on the Metal Centers of Cytochrome c
Oxidase," Jeff Gelles, David F. Blair, Craig T. Martin, Hsin Wang,
and Sunney I. Chan, in Frontiers in Biochemical and Biophysical
Studies of Proteins and Membranes, Teh-Yung Liu, Shunpei
Sakakibara, Alan N. Schechter, Kunio Yagi, Haruaki Yajima, and Kerry
T. Yasunobu, eds., Elsevier, New York, 259-277, 1983.
"The Structure of the Metal Centers in Cytochrome c
Oxidase," Sunney I. Chan, Craig T. Martin, Hsin Wang, Gary W.
Brudvig, and Tom H. Stevens, in The Coordination Chemistry of
Metalloenzymes, I. Bertini, R. S. Drago, and C. Luchinat, eds.,
D. Reidel Pub. Co., Boston, 313-328, 1983.
"A Resonance Raman Investigation of Perturbed States of Tree
and Fungal Laccase," D. F. Blair, G. W. Campbell, V. Lum, C. T.
Martin, H. B. Gray, B. G. Malmström, and Sunney I. Chan,
J. Inorg. Biochem. 19, 65-73, 1983.
"The Metal Centers of Cytochrome c Oxidase: Structures
and Interactions," David F. Blair, Craig T. Martin, Jeff Gelles, Hsin
Wang, Gary W. Brudvig, Tom H. Stevens, and Sunney I. Chan, Chemica
Scripta 21, 43-53, 1983.
"The Nature and Distribution of the Metal Centers in Cytochrome
c Oxidase," Sunney I. Chan, Gary W. Brudvig, Craig T. Martin,
and Tom H. Stevens, in Electron Transport and Oxygen
Utilization, Chien Ho, ed., Elsevier, Amsterdam, 171-177, 1982.
"The Nature of CuA in Cytochrome c Oxidase," Tom H. Stevens, Craig T. Martin, Hsin Wang, Gary W. Brudvig, Charles P. Scholes, and Sunney I. Chan, J. Biol. Chem. 257,
"Reactions of Nitric Oxide with Tree and Fungal Laccase," Craig T. Martin, Randall H. Morse, Robert M. Kanne, Harry B. Gray, Bo G. Malmström, and Sunney I. Chan, Biochemistry 20, 5147-5155,
Software Review of (curve fitting software) "pro Fit 5.01 for the Mac" J. Am. Chem. Soc. 119(30), 7171-7172, 1997.
Book Review: "DNA-Protein Interactions: Principles and Protocols, 2nd. Edition," Edited by Tom Moss, Craig T. Martin, ChemBioChem 4(6), 546, 2003.
||FASEB Summer Conference on Initiation of Transcription in Prokaryotes, Saxtons River, VT
||Minority Biomedical Research Program, Cayey University College, Cayey, Puerto Rico
|Nov xx, 2006
||FASEB Summer Conference on Initiation of Transcription in Prokaryotes, Saxtons River, VT
|Nov 6, 2006
||The Pennsylvania State University, University Park
|Nov 16, 2005
||University of Connecticut
|Apr 29, 2005
||University of California at Santa Barbara
|Nov 10, 2004
|May 1, 2003
||Case Western Reserve University
|April 30, 2003
|Nov 12, 2002
||University at Albany
|March 18, 2002
||Dept of Biochemistry and Molecular Biology, Pennsylvania State Medical Center, Hershey, PA
|March 16, 2002
||MCB Annual Retreat, University of Massachusetts, Amherst, MA
|February 23, 2002
||Annual Meeting of the Biophysical Society, San Francisco, CA, Fluorescence Subgroup Meeting
||FASEB Summer Conference on Initiation of Transcription in Prokaryotes, Saxtons River, VT
|November 10, 1999
||Department of Chemistry, University of Massachusetts at Dartmouth
|May 27, 1999
||Centro de Biologia Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Spain
|May 17, 1999
||Institut Pasteur, Departement de Biologie Moleculaire, Paris, France
|April 15, 1999
||Department of Biology, University of Indiana, Bloomington, IN
|April 2, 1999
||Department of Chemistry, Wesleyan University, Middletown, CT
|April 23, 1998
||Department of Chemistry, State University of New York at Buffalo
||Minority Biomedical Research Program, Cayey University College, Cayey, Puerto Rico
|February 3, 1998
||Department of Immunology and Microbiology, State University of New York at Brooklyn
|January 22, 1998
||Department of Chemistry, University of Pennsylvania
|October 5, 1997
||Department of Chemistry, California Institute of Technology
|April 28, 1997
||Program in Molecular & Cellular Biology, University of Massachusetts
|April 17, 1997
||Department of Biochemistry, Louisiana State Medical Center
Craig T. Martin