Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters

Martin, Craig T.; Coleman, Joseph E.

doi:10.1021/bi00384a006

Cited by 135 publications

(167 citation statements)

References 25 publications

Supporting

Mentioning

149

Contrasting

Order By: Relevance

“…All promoter fragments from P-17 to P-27 as well as mP-22 and mP1.1B-22 showed similar amounts of short abortive products. This is in agreement with equal amounts of 5-nt RNA (full-length) produced from Ϫ19 and Ϫ17 truncated promoters, as reported earlier (30). Interestingly, the amounts of long abortive products from P-17 to P-27 promoter fragments exhibited a direct dependence on the specific upstream promoter length.…”

Section: The Use Of [␥-supporting

confidence: 91%

“…However, no significant differences were noted in the same study for other promoters with incremental extensions up to Ϫ25 position even with those containing the same GC pair at the Ϫ18 position. Promoters extended upstream to Ϫ19 position (with AT pairs) or much longer (up to 3-kb linear plasmid) have been shown to produce the same amounts of 5-or 24-nt transcript as the minimal Ϫ17 truncated promoter (29,30). We have measured the kinetics of 2-19-nt (full-length) RNA synthesis on P-17, P-21, P-22, P-27, mP-22, and mP1.1B-22 promoter fragments.…”

Section: Kinetics Of Promoter Binding and Dissociation-mentioning

confidence: 99%

See 1 more Smart Citation

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Tang¹,

Bandwar²,

Patel³

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Bacteriophage T7 promoters contain a consensus sequence from ؊17 to ؉6 relative to the transcription start site, ؉1. In addition, the strong class III promoters are characterized by an extended ATrich region upstream of ؊17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of ؊17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between ؊17 and ؊27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond ؊22. Similarly, promoters in which the AT-rich region from ؊17 to ؊22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of ؊17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.Bacteriophage T7 RNA polymerase (RNAP) 2 and T7 promoters constitute a model system for studying the protein-DNA interactions that occur during transcription as well as to understand the basic catalytic mechanism of DNA transcription. The 99-kDa single subunit enzyme of phage T7 shares many functional characteristics of transcription catalysis with multisubunit RNAPs despite lacking proteins structural similarities. T7 RNAP, without the assistance of accessory proteins, is capable of catalyzing all the fundamental transcription activities. The various crystal structures of T7 RNAP-promoter DNA complex provide deeper insights into the interactions that occur with the promoter as well as conformational changes in T7 RNAP that occur during promoter clearance (1-4). The structure of T7 RNAP bound to a minimal promoter fragment shows that the specific recognition of T7 promoter involves both base-specific and nonspecific contacts with the 13 conserved promoter base pairs from Ϫ17 to Ϫ5. These include upstream contacts in the major groove of the specificity region of the promoter (Ϫ11 to Ϫ5) and in the minor groove of the AT-rich region of the promoter (Ϫ17 to Ϫ13) (1, 2). The base pairs from Ϫ4 to ϳϩ2 that include the initiation site (ϩ1) are melted, and the single-stranded template DNA is plunged ...

show abstract

Section: The Use Of [␥-supporting

confidence: 91%

Section: Kinetics Of Promoter Binding and Dissociation-mentioning

confidence: 99%

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Tang¹,

Bandwar²,

Patel³

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The phage enzymes are among the simplest RNA polymerases known, as no accessory proteins are necessary for specific initiation, elongation, or termination of transcription (2,3). The 17 promoters of bacteriophage T7 direct specific initiation of RNA synthesis that occurs in a rapid and processive manner (4,5). Due to their simplicity these enzymes serve as model systems to understand, in depth, the mechanisms of transcription initiation, elongation, or termination.…”

mentioning

confidence: 99%

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

Jia

Kumar

Patel

1996

Journal of Biological Chemistry

113

120

View full text Add to dashboard Cite

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 ED a complex rapidly with an equilibrium association constant K 1 . The ED a complex then undergoes a conformational change to ED b , 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 ED a to ED b 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, ⌽10 and ⌽3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, k on (K 1 k 2 ), and the dissociation rate constant, k off (k ؊2 ), and thermodynamics, the equilibrium constants of the two steps (K 1 and k 2 /k ؊2 ) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower k on , the 4-fold higher k off , and the 2-5-fold weaker equilibrium interactions together make ⌽3.8 a weaker promoter relative to ⌽10.Bacteriophage T7 RNA polymerase is a 98-kDa single subunit polymerase that catalyzes synthesis of RNA complementary in sequence to the template DNA (1). The phage enzymes are among the simplest RNA polymerases known, as no accessory proteins are necessary for specific initiation, elongation, or termination of transcription (2, 3). The 17 promoters of bacteriophage T7 direct specific initiation of RNA synthesis that occurs in a rapid and processive manner (4, 5). Due to their simplicity these enzymes serve as model systems to understand, in depth, the mechanisms of transcription initiation, elongation, or termination.Initiation of transcription occurs by recognition and binding of the RNA polymerase to a promoter DNA sequence. This event is recognized as one of the important steps at which transcription and gene expression is regulated. The 17 bacteriophage T7 promoters share consensus sequence from Ϫ17 to ϩ6 position relative to the transcription start site at ϩ1 (6). The class III gene promoters of T7 are absolutely conserved in DNA sequence, whereas the class II gene promoters diff...

show abstract

“…A 17-nucleotide 5' promoter sequence directs efficient initiation at a unique position on the DNA template (2). While the transcription generally initiates with a nucleoside triphosphate (NTP), extension of a mono-, di-, or trinucleotide primer can occur if it is present in high concentration and has a sequence appropriate to initiate at or near the normal start site (3).…”

mentioning

confidence: 99%

RNA template-directed RNA synthesis by T7 RNA polymerase.

Cazenave

Uhlenbeck

1994

Proc. Natl. Acad. Sci. U.S.A.

139

130

View full text Add to dashboard Cite

In an attempt to synthesize an oligoribonucleotide by run-off transcription by bacteriophage 17 RNA polymerase, a major t ript was produced that was much longer than expected. Analysis ofthe reaction indicated that the product resulted from initial DNA-directed run-off t iption foflowed by RNA templatedrected RNA synthe. This reaction occurred because the RNA made from the DNA template displayed self-complementarit at Its 3' end and therefore could form an iatra-or intermolecular primed template. In reactions contining only an RNA template, the rate of incorporation of NTPs was quite comparable to DNAdependent transcription RNA template-irected RNA synthesis has been found to occur with a great number of oligoribonucleotides, even with primed templates that are only marginally stable. In one instance, we observed a multisep e on reaction converting the oligonucleotide into a final product longer than twice Its original length. Presumably, such a process could have generated some of the RNAs found to be efflclently replicated by ¶7 RNA polymerase.The RNA polymerases from bacteriophage T3, T7, and SP6 have been widely used to prepare RNAs for biochemical and biophysical studies (1). A 17-nucleotide 5' promoter sequence directs efficient initiation at a unique position on the DNA template (2). While the transcription generally initiates with a nucleoside triphosphate (NTP), extension of a mono-, di-, or trinucleotide primer can occur if it is present in high concentration and has a sequence appropriate to initiate at or near the normal start site (3). In the absence of a promoter, bacteriophage polymerases can bind to the end of a DNA fragment and, with much lower efficiency, produce an "endto-end" transcription product (4).In most in vitro transcription reactions, the expected run-off RNA corresponding precisely to the template sequence is not the only product obtained. A number of abortive initiation products of 2-8 nucleotides are generally produced in substantial amounts (1,5 pH 8.1 (at 37r()/10 mM MgC12/1 mM spermidine/5 mM dithiothreitol/0.01% Triton X-100 for 1 hr at 3rc. Nonradioactive RNA was obtained similarly except that each NTP was 4 mM, MgCl2 concentration was raised to 25 mM, the final volume was 5 ml, and the incubation was for 3 hr at 370C. Transcripts were purified by electrophoresis in a 1.5-mm-thick 20%o polyacrylamide sequencing gel, located by autoradiography or UV shadowing, eluted by crushing in 0.25 M ammonium acetate/10 mM Tris-HCl, pH 8.0/1 mM EDTA, and concentrated by DEAE chromatography and ethanol precipitation. RNA was dissolved in sterile water and stored at -20oC.Oligomers 1A (GAAUGUCGGUCG), 3 (GCUAGCAU-CC), 4 (GAAUGUGGAUGC), and 5 (CGGUCG) were chemically synthesized on an Applied Biosystems DNA synthesizer using phosphoramidite chemistry (10), deprotected, and purified by gel electrophoresis. Concentrations of oligomers were determined by their absorbance at 260 nm, with extinction coefficients calculated from their base composition and sequence (11).5'-End-labeled oligo...

show abstract

Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters

Cited by 135 publications

References 25 publications

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

RNA template-directed RNA synthesis by T7 RNA polymerase.

Contact Info

Product

Resources

About