PubMed · 9204424
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
A Siwkowski. 1997. T7 transcript length determination using enzymatic RNA sequencing.. https://doi.org/10.1385/0-89603-389-9%3A91
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.
To make messenger RNA transcripts, bacteriophage T7 RNA polymerase (T7 RNAP) undergoes a transition from an initiation phase, which only makes short RNA fragments, to a stable elongation phase. We have determined at 2.1 angstrom resolution the crystal structure of a T7 RNAP elongation complex with 30 base pairs of duplex DNA containing a "transcription bubble" interacting with a 17-nucleotide RNA transcript. The transition from an initiation to an elongation complex is accompanied by a major refolding of the amino-terminal 300 residues. This results in loss of the promoter binding site, facilitating promoter clearance, and creates a tunnel that surrounds the RNA transcript after it peels off a seven-base pair heteroduplex. Formation of the exit tunnel explains the enhanced processivity of the elongation complex. Downstream duplex DNA binds to the fingers domain, and its orientation relative to upstream DNA in the initiation complex implies an unwinding that could facilitate formation of the open promoter complex.
The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over approximately 24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme. Ocr can also bind to the component subunits of EcoKI. Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change. This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme. This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.