Recognition Sites of 3′-OH Group by T7 RNA Polymerase and Its Application to Transcriptional Sequencing

Abstract

When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing." T7 RNAP1 is a single 99-kDa polypeptide containing in its catalytic domain a highly specific promoter recognition site and a nascent RNA binding domain. T7 RNAP displays a stringent specificity for T7 promoter to initiate transcription. In T7 phage infection and the lytic cycle, T7 RNAP transcribes class II and III of the T7 phage genome (1). Based on three-dimensional structure analysis and mutagenesis of the Klenow fragment, the protein critical regions and structural motifs for polymerase activity have been suggested. Additionally, from sequence homologies of the Klenow fragment, Taq DNA polymerase, and T7 DNA polymerase, structural similarities have also been suggested in motifs A (532-555 aa), B (625-652 aa), and C (805-818 aa) (2). From mutagenesis analysis, the critical sites of primer recognition, metal ion binding, rNTP binding, and polymerase activity have been determined; however these do not necessarily coincide with the homologous motifs. Phenylalanine and tyrosine have been reported to play important roles in discriminating 2Ј-and 3Ј-OH groups in Escherichia coli DNA polymerase I (3, 4) and Taq DNA polymerase (5). These findings indicate that the aromatic ring of phenylalanine or tyrosine is needed to discriminate the 2Ј-and 3Ј-OH groups. Using mutant characterization and three-dimensional structure analysis (6), the catalytic domain of T7 RNAP was separated into the T7 promoter binding region (674 -752 aa), Mg 2ϩ binding sites (Asp-537, Asp-812), and essential regions of polymerase activity (motif A, motif B, motif C). To analyze the mechanism of the polymerase reaction, the interaction sites for the 2Ј-and 3Ј-OH group have been extensively examined (3-5).Recently, Tyr-639 in the active site has been reported to be a discrimination site for 2Ј-OH groups in T7 RNAP (7). The Y639F mutant retains DNA and RNA polymerase activities but cannot discriminate rNTP from 2Ј-dNTPs. To further determine the mechanisms of the polymerase reaction, the 3Ј-OH discrimination site in the elongation step needed to be identified. However, details of the ...

Automated Filtration-Based High-Throughput Plasmid Preparation System

“…As an additional step, we examined whether or not the prepared plasmids were useful for our previously described transcriptional sequencing method (Sasaki et al 1998a,b;Izawa et al 1998). Transcriptional sequencing is based on RNA transcription from a promoter with T7 RNA polymerase without the need for sequencing primers.…”

“…DNAs were dissolved in TE buffer, precipitated with ethanol, and, then, sequenced. Sequencing was as described previously (Sasaki et al 1998a,b;Izawa et al 1998). An ABI 377 DNA sequencer was used to anlayze the sequence.…”

“…The dried DNAs were dissolved in 10 µl of distilled water and then applied to a spin column for desalting. Transcriptional sequencing was performed by a previously reported method (Izawa et al 1998;Sasaki et al 1998a,b). The residual free dye terminator was removed as described above.…”

“…Recently, we developed a transcriptional sequencing technique based on the chain termination method and using T7 RNA polymerase (Sasaki et al 1998a,b;Izawa et al 1998). For this method, T7 RNA polymerase transcribes the DNA template from the T7 promoter and produces RNA chain termination products.…”

RIKEN Integrated Sequence Analysis (RISA) System---384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

“…Direct sequencing reactions are carried out in 20 µL containing 10 ng of unpurified PCR products, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl 2 , 5 mM DTT, 2 mM spermidine-(HCl) 3 , 2 mM 1,8 diamino-octane, 25 mM NaCl, 0.4 mM MnCl 2 , and a transcriptional sequencing kit (TS kit) based on mutant T7 RNA Polymerase (Izawa et al 1998;Sasaki et al 1998a,b; the TS kit is being prepared for marketing). These reaction mixtures were incubated at 37°C for 60 min.…”