Transition from initiation to promoter proximal pausing requires the CTD of RNA polymerase II.
The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II Delta5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo. Here, we demonstrate that in the context of chromatin, Pol II Delta5 can bind to the c-myc promoter with the same efficiency as wild type Pol II. However, Pol II Delta5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged Delta5 or wildtype Pol II revealed a single, highly mobile Pol II Delta5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex.
Lux
C
C
Institute of Clinical Molecular Biology and Tumour Genetics, GSF-National Research Center for Environment and Health, Marchioninistrasse 25, D-81377 Munich, Germany.
Albiez
H
H
Chapman
R D
RD
Heidinger
M
M
Meininghaus
M
M
Brack-Werner
R
R
Lang
A
A
Ziegler
M
M
Cremer
T
T
Eick
D
D
D001665:Binding Sites; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D016384:Consensus Sequence; D036681:Fluorescence Recovery After Photobleaching; D016259:Genes, myc; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D014158:Transcription, Genetic
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II