Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

FEBS Letters

Herdewijn, Piet²  Tunitskaya, Vera L¹  Ermolinsky, Boris S¹  Rusakova, Ekaterina E¹  Efimtseva, Ekaterina V¹  Van Aerschot, Arthur²  Memelova, Lyudmila V¹  Kochetkov, Sergey N¹  Mikhailov, Sergey N¹ 

[1] Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, 117984 Moscow, Russia;Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

关键词: T7 RNA polymerase;    Transcription;    Promoter;    Oligonucleotide;    Dialdehyde group;    Affinity labeling;    T7 RNAP;    bacteriophage T7 RNA polymerase;    ON;    oligonucleotide;    aa;    amino acid;   

DOI  :  10.1016/S0014-5793(98)01625-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Oligonucleotides of a novel type containing 2′-O-β-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein-nucleic acid interactions.

【 授权许可】

Unknown