Rapid Mutagenesis and Purification of Phage RNA Polymerases

He, Biao; Rong, Mingqing; Lyakhov, D. L.; Gartenstein, Heidi; Diaz, George A.; Castagna, Ray; McAllister, William T.; Durbin, Russell K.

doi:10.1006/prep.1996.0663

Cited by 141 publications

(130 citation statements)

References 3 publications

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“…T7 RNA Polymerase-T7 RNA polymerase with a histidine tag was prepared from Escherichia coli strain BL21 carrying the plasmid HB161 (kindly supplied by W. T. McAllister), in which RNA polymerase is expressed under inducible control of the lac UV5 promoter (13). The T7 RNA polymerase P266L mutant was produced by QuikChange mutagenesis (Agilent Technologies, Santa Clara, CA).…”

Section: Methodsmentioning

confidence: 99%

New Insights into the Mechanism of Initial Transcription

Ramírez-Tapia

Martin

2012

Journal of Biological Chemistry

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Background: RNA polymerases must couple the energetics of nucleotide addition to the timed release of promoter contacts. Results: A mutant that aborts less during initial transcription releases the promoter at longer RNA lengths. Conclusion: Hybrid growth remodels the protein to disrupt promoter contacts; the protein, in turn, pushes back on the hybrid, leading to abortive instability. Significance: The model extends to multisubunit RNA polymerases.

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Section: Methodsmentioning

confidence: 99%

New Insights into the Mechanism of Initial Transcription

Ramírez-Tapia

Martin

2012

Journal of Biological Chemistry

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“…Nonetheless, the mechanism of T7 RNAP transcription shares significant similarities with multisubunit RNA polymerases, and it is likely that fundamental aspects of transcription are maintained between T7 RNAP and multisubunit RNA polymerases (24,25). To take advantage of its simplicity to study the functions of the phosphorylated CTD in transcription and RNA processing, we constructed a plasmid that expresses His-tagged T7 RNAP (26) fused with the mouse Pol II CTD. In an initial experiment, T7 RNAP fused to the CTD at its C terminus and purified from Escherichia coli had no detectable transcription activity (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…The plasmid (pBH161)-expressing His-tagged T7 RNAP was a gift from W. McAllister (State University of New York Downstate Medical Center, Brooklyn, NY) (26). For expression of CTD-T7 RNAP, which consists of the His-tagged CTD from mouse Pol II fused to the N terminus of T7 RNAP, the mouse CTD generated by PCR was inserted into pBH161 with XhoI sites (pBH161-CTD).…”

Section: Methodsmentioning

confidence: 99%

Human capping enzyme promotes formation of transcriptional R loops in vitro

Kaneko

Chang²,

Shatkin³

et al. 2007

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

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Cap formation is the first step of pre-mRNA processing in eukaryotic cells. Immediately after transcription initiation, capping enzyme (CE) is recruited to RNA polymerase II (Pol II) by the phosphorylated carboxyl-terminal domain of the Pol II largest subunit (CTD), allowing cotranscriptional capping of the nascent premRNA. Recent studies have indicated that CE affects transcription elongation and have suggested a checkpoint model in which cotranscriptional capping is a necessary step for the early phase of transcription. To investigate further the role of the CTD in linking transcription and processing, we generated a fusion protein of the mouse CTD with T7 RNA polymerase (CTD-T7 RNAP). Unexpectedly, in vitro transcription assays with CTD-T7 RNAP showed that CE promotes formation of DNA⅐RNA hybrids or R loops. Significantly, phosphorylation of the CTD was required for CE-dependent R-loop formation (RLF), consistent with a critical role for the CTD in CE recruitment to the transcription complex. The guanylyltransferase domain was necessary and sufficient for RLF, but catalytic activity was not required. In vitro assays with appropriate synthetic substrates indicate that CE can promote RLF independent of transcription. ASF/SF2, a splicing factor known to prevent RLF, and GTP, which affects CE conformation, antagonized CE-dependent RLF. Our findings suggest that CE can play a direct role in transcription by modulating displacement of nascent RNA during transcription.RNA displacement ͉ RNA polymerase II ͉ RNA processing R NA polymerase II (Pol II) plays a critical role not only in the transcription of mRNA precursors, but also in their subsequent processing. The best characterized example is cotranscriptional cap formation, which is achieved by the physical interaction of capping enzymes (CEs) with the phosphorylated carboxyl-terminal domain of the Pol II largest subunit (CTD) (1-5). The CTD, composed mainly of a repeated heptapeptide motif, YSPTSPS, is extensively phosphorylated, and its regulation is tightly controlled during the transcription cycle (6-8). Phosphorylation of serine-5 in the heptad repeats, by Cdk7 in human and Kin28 in yeast, occurs shortly after transcription initiation and is required for cotranscriptional recruitment of CE (9, 10).The formation of the 5Ј-terminal m7G(5Ј)ppp(5Ј)N cap is the first step of pre-mRNA processing and involves a series of three enzymatic reactions. RNA triphosphatase (RTPase) removes a phosphate from the 5Ј end of the nascent transcript, RNA guanylyltransferase (GTase) transfers GMP from GTP to the diphosphate RNA terminus, and RNA (guanine-N7) methyltransferase (MTase) adds a methyl group (11). In budding yeast, capping is carried out by three polypeptides that are encoded separately (12). Cet1 (RTPase) interacts with Ceg1 (GTase), and this interaction is critical for stimulating Ceg1 activity (13). Abd1 (MTase) and Ceg1 bind directly to the phosphorylated CTD (2, 3). Mammalian CEs consist of two components, a bifunctional CE (with an N-terminal RTPase domain and C-t...

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“…Plasmids encoding tRNA Leu (UAA) isoacceptor sequences from A. tumefaciens C58 or E. coli in front of T7 polymerase promoter sequences were purified from E. coli DH5-a cells before digestion overnight at 60 1C with BstNI restriction enzyme to linearize the plasmid DNA. The transcription reaction was performed using template DNA (450 mg), 9 mM T7 RNA polymerase 35 , 1 U ml À 1 RNAse inhibitor, 40 mM Tris-HCl (pH 8.0), 25 mM MgCl 2 , 40 mM DTT, 0.1% Triton X-100, 1 mM spermidine and 2 mM rNTPs. The reaction mix was incubated at 37 1C overnight before quenching with 50 mM EDTA followed by DNase I digestion for 1 h to remove template DNA.…”

Section: Methodsmentioning

confidence: 99%

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

et al. 2013

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Leucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA Leu for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3 0 -end. Our findings reveal an intriguing tRNAdependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design.

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Rapid Mutagenesis and Purification of Phage RNA Polymerases

Cited by 141 publications

References 3 publications

New Insights into the Mechanism of Initial Transcription

New Insights into the Mechanism of Initial Transcription

Human capping enzyme promotes formation of transcriptional R loops in vitro

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

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