Open AccessPhoto-Cross-Linking of a Purified Preinitiation Complex Reveals Central Roles for the RNA Polymerase II Mobile Clamp and TFIIE in Initiation Mechanisms
Author(s) -
Diane Forget,
Marie-France Langelier,
Cynthia Thérien,
Vincent Q. Trinh,
Benoit Coulombe
Publication year - 2004
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.24.3.1122-1131.2004
Subject(s) - transcription factor ii a , transcription factor ii e , transcription preinitiation complex , biology , transcription factor ii f , transcription factor ii b , microbiology and biotechnology , rna polymerase ii , transcription bubble , general transcription factor , promoter , genetics , gene , gene expression
The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIE alpha closely approaches promoter DNA at nucleotide -10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIE alpha is obtained when the DNA template is artificially premelted in the -11/-1 region, suggesting that TFIIE alpha facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.