Stable transcription-elongation complexes consisting of T7 RNA polymerase (molecular mass 99 kDa) in association with a nucleic acid scaffold consisting of an 8 bp RNA-DNA hybrid and 10 bp of downstream DNA were assembled and crystallized by the sitting-drop vapour-diffusion technique under near-physiological conditions. The crystals diffract beyond 2.6 A resolution and belong to space group P1...

Escherichia coli RNA polymerase (EC 2.7.7.6), bound in a tight complex at an early T7 promoter, protects 41 to 43 base pairs of DNA from digestion by DNase. I. The protected DNA fragment contains both the binding site for RNA polymerase and the mRNA initiation point for the promoter. The sequence of the DNA fragment and the sequence of the mRNA that it codes for are presented here. A seven-base...

Protein enzymes frequently recruit small molecule coenzymes to perform a variety of biochemical reactions. While the catalytic activities of RNA have been expanding rapidly, a similar strategy for RNA to utilize coenzymes and to increase its functional capabilities has yet to be demonstrated. A general in vitro transcription procedure has been developed to efficiently prepare RNA with coenzymes...

Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A com...

Molecular information theory was used to create sequence logos and promoter models for eight phages of the T7 group: T7, phiA1122, T3, phiYeO3-12, SP6, K1-5, gh-1 and K11. When these models were used to scan the corresponding genomes, a significant gap in the individual information distribution was observed between functional promoter sites and other sequences, suggesting that the models can be...

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a n...

The structures of T7 RNA polymerase (T7 RNAP) captured in the initiation and elongation phases of transcription, that of phi29 DNA polymerase bound to a primer protein and those of the multisubunit RNAPs bound to initiating factors provide insights into how these proteins can initiate RNA synthesis and synthesize 6-10 nucleotides while remaining bound to the site of initiation. Structural insig...

We have constructed a plasmid expressing E. coli M1 RNA, the catalytic RNA subunit of ribonuclease P, under the control of a phage T7 promoter. The active M1 RNA species synthesized in vitro by T7 RNA polymerase from this vector was reacted with the tRNA(Gln) - tRNA(Leu) precursor RNA (Band K) encoded by phage T4. Only the tRNA(Leu) moiety of this dimeric precursor RNA contains the 3' terminal ...