Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products.
We have explored the effects of a variety of structural and sequence changes in the initiation region of the phage T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region. Although the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and stabilization of the initiation complex. At promoters in which the integrity of T strand is interrupted by nicks or gaps between -5 and -2 the enzyme continues to initiate predominately at +1. However, under these conditions there is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nascent transcript), and a defect in displacement of the RNA. We propose that unlinking the binding and initiation regions of the T strand changes the manner in which this strand is retained in the abortive complex, reducing or eliminating the need to pack or "scrunch" the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation.
Jiang
M
M
Morse Institute of Molecular Genetics, Department of Microbiology and Immunology, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, USA.
Rong
M
M
Martin
C
C
McAllister
W T
WT
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005814:Genes, Viral / Q000235:genetics; D008957:Models, Genetic; D016254:Mutagenesis, Insertional / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011068:Poly G / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012313:RNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D013698:Templates, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins
D009692:Nucleic Acid Heteroduplexes; D014764:Viral Proteins; D011068:Poly G; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases