The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR...
In contrast to the phage-dependent variation of sequences in the binding region, the initiation region sequences, especially from −7 to +1, are virtually identical for all phage promoters. Binding of a polymerase to its promoter results in melting of the dsDNA in the initiation region, as...
Here, we use 5′RACE-Seq to screen a randomized initially transcribed region of the T7 promoter for cross-talk with transcriptional activity. We reveal that sequences from position +4 to +8 downstream of the transcription start site affect T7 promoter activity over a 5-fold range, and...
coli RNAP and one T7 promoter lie on this internalized segment, and transcription from these promoters normally pulls the genome into the cell. In the absence of T7 RNAP, E. coli RNAP internalizes the entire T7 genome at a constant 40 bp/s at 30 °C, approximately the same rate of...
In contrast to the phage-dependent variation of sequences in the binding region, the initiation region sequences, especially from −7 to +1, are virtually identical for all phage promoters. Binding of a polymerase to its promoter results in melting of the dsDNA in the initiation region, as...
We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family...
Promoter Structure, Recognition, and Opening The T7 promoter is 23 bp in length and has a tri-partite structure (Figure 4). The −17 to −6 sequence is important for specific binding of the polymerase via interactions between residues 746, 748, 756, and 758 and the −7 to −11 ...
The DNA template consists of the T7 promoter sequence followed by the RNA of interest. 2 Transcription with T7 RNA polymerase is performed in 40 mM Tris–HCl, pH 8.1, 1 mM spermidine, 50 μg/ml BSA, 5–10 μCi [α-32P]ATP, 14 mM MgCl2, 60 μg/ml plasmid DNA, and 40 μg/ml...
2. The DNA sequence of claim 1 in which only nucleotide residue 639 has been deleted. 3. The DNA sequence of claim 1 in which the promoter is a lac or lambda PLpromoter. 4. The DNA sequence of claim 3 in which the promoter is a lac UV5 promoter. ...
The coding sequence for the desired RNA or protein (referred to as the target RNA or protein) is typically placed in a plasmid under control of a T7 promoter, that is, a promoter recognized specifically by T7 RNA polymerase. In the absence of an inducer for the lacUV5 promoter, little ...