Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347

King, Garry C.; Martin, Craig T.; Pham, Thang; Coleman, Joseph E.

doi:10.1021/bi00349a006

Cited by 95 publications

(84 citation statements)

References 17 publications

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“…The purified enzyme was stored at Ϫ80°C in 20 mM sodium phosphate, pH 7.7, 1 mM trisodium EDTA, 1 mM dithiothreitol, 100 mM sodium chloride, and 50% (v/v) glycerol. The enzyme concentration was calculated from its absorbance at 280 nm and with a molar extinction coefficient of 1.4 ϫ 10 5 M Ϫ1 cm Ϫ1 (16). T7 lysozyme was purified according to a reported procedure (10).…”

Section: Methodsmentioning

confidence: 99%

T7 Lysozyme Represses T7 RNA Polymerase Transcription by Destabilizing the Open Complex during Initiation

Stano

Patel

2004

Journal of Biological Chemistry

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. This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP K d is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from ؊4 to ؉2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2-to 5-mer that delays the transition from the initiation to the elongation phase.The regulation of transcription in bacteriophage T7 RNA polymerase (RNAP) 1 as in all RNA polymerases is dependent on the efficiency of each step of transcription, such as binding promoter DNA, binding nucleotides, overcoming abortive synthesis, RNA synthesis, and termination. Each of these steps is therefore a potential target for regulation. T7 transcription regulation begins with the gradual entry of the phage DNA into a host Escherichia coli cell (1) followed by the ability of the T7 RNAP, without the aid of auxiliary factors, to recognize and transcribe the phage promoters (2, 3). The phage then makes T7 lysozyme, which represses transcription by T7 RNA polymerase. Unlike the LacI or the LexA repressors, which sterically block promoters from their polymerases, T7 lysozyme binds the T7 RNAP, not the DNA, forming a tertiary complex with the polymerase and DNA (4 -7). T7 lysozyme binds to the T7 RNAP at a site distal to the polymerase active site as confirmed by both biochemical data and a crystal structure of the T7 RNAP-T7 lysozyme complex (8 -10).Previous studies have shown that T7 lysozyme inhibits transcription initiation and promoter clearance but not elongation (6, 7). However, the exact mechanism by which these processes are inhibited is not known. We have therefore investigated T7 lysozyme inhibition under pre-steady state conditions enabling us to more fully dissect the steps that are altered during transcription. Transcription initiation occurs with a minimum of three steps (Reaction 1 A stopped flow kinetic study of DNA binding showed that T7 lysozyme has a very small effect on the observed rate of formation of the preinitiation open complex. In the presence of T7 lysozyme, the initial RNA synthesis rate is slow even with saturating initiating nucleotide. The 2-AP fluorescence of the DNA indicates that the promoter in the initiation complex is not fully open. This is consistent with the results that T7 lysozyme affects the rate of initiation on a duplex promoter but not on a permanently open promoter DNA or a promoter DNA with even a single mismatch in the initiation region. The destabilizatio...

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Section: Methodsmentioning

confidence: 99%

T7 Lysozyme Represses T7 RNA Polymerase Transcription by Destabilizing the Open Complex during Initiation

Stano

Patel

2004

Journal of Biological Chemistry

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“…The enzyme was purified, and its concentration was determined (⑀ 280 ϭ 1.4 ϫ 10 5 M Ϫ1 cm Ϫ1 ) as described previously (14). Purity of the enzyme was verified by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

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

Kuzmine¹,

Gottlieb²,

Martin³

2003

Journal of Biological Chemistry

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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 17 T7 RNA polymerase promoters in the phage genome, 15 begin with guanine. In this work, we demonstrate that the purine N-7 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 O-6 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.The initiation of transcription imposes some unique mechanistic requirements on an RNA polymerase. In contrast to events occurring during elongation, at the initial step of transcription initiation, two substrate nucleoside triphosphate molecules must position accurately in the active site. Clearly a part of the binding energetics is derived from Watson-Crick interactions between the incoming bases and those in the template strand of DNA, but just as clearly, base pairing interactions are not sufficient to provide the binding energetics required for full function. Indeed, a single Watson-Crick base pair is unstable in solution (1).It is understood that for the Watson-Crick placement of the elongating nucleotide (position ϩ2 at initiation), additional energy for binding of the nucleotide comes from interactions between its triphosphate, magnesium, and protein functional groups (2-5). This interaction would not be expected to be important in binding the initiating (ϩ1) nucleotide, and it has been shown in the T7 system that guanosine monophosphate and even the nucleoside guanosine have K m values comparable to or lower than that of the triphosphate GTP (6). Some additional interaction(s) must be at play.Most RNA polymerases show some preference for the initial base of the transcript. Escherichia coli RNA polymerase promoters often initiate with ATP, although some promoters begin with other NTPs at the first position in the transcript (7,8). Of the 17 phage RNA polymerase promoters in the T7 genome, the canonical ϩ1 position of 15 begins with GTP, while two promoters begin with ATP (9). Recent studies have demonstrated that ...

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“…Studier), in which RNA polymerase is expressed under inducible control of the lac UV5 promoter (45). The enzyme was purified and the concentration was determined (ε 280 = 1.4 × 10 5 M −1 cm −1 ) as described previously (46). Purity of the enzyme (>95%) was verified by SDS-denaturing polyacrylamide gel electrophoresis.…”

Section: T7 Rna Polymerasementioning

confidence: 99%

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

et al. 2007

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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 Cterminal 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.

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Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347

Cited by 95 publications

References 17 publications

T7 Lysozyme Represses T7 RNA Polymerase Transcription by Destabilizing the Open Complex during Initiation

T7 Lysozyme Represses T7 RNA Polymerase Transcription by Destabilizing the Open Complex during Initiation

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

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

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