Point Contacts of T7 RNA Polymerase in the Promoter Complex, as Determined with Phosphate-Activated Oligonucleotide Derivatives

• Published in: Molecular biology (New York) Vol. 36; no. 4; pp. 543 - 550
• Main Authors: Filippova, S E, Ivanovskaya, M G, Romanova, E A, Tunitskaya, V L, Kochetkov, S N
• Format: Journal Article
• Language: English
• Published: New York Springer Nature B.V 01-07-2002
• Subjects: Phosphates; RNA polymerase
• ISSN: 0026-8933; 1608-3245
• DOI: 10.1023/A:1019864729819

The contacts between phosphate groups of promoter DNA and Lys or His of T7 RNA polymerase (Pol) in the Pol-promoter complex were studied with single- and double-stranded oligonucleotides corresponding to the T7 promoter consensus and containing activated phosphate groups at position +1, +2, or -14 relative to the transcription start. To obtain reactive groups, terminal phosphates were modified with N-hydroxybenzotriazole (HOBT), and internucleotide phosphates were replaced with a trisubstituted pyrophosphate (TSP). The resulting derivatives produced covalent complexes with T7 Pol. Covalent bonding involved His in the case of TSP at position +1 or HOBT at position +1 or -14, and Lys in the case of TSP at position -14.[PUBLICATION ABSTRACT]