RESUMO
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.
Assuntos
Regiões Promotoras Genéticas , RNA Polimerase II/química , Transcrição Gênica , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Sequência Consenso , Recuperação de Fluorescência Após Fotodegradação , Genes myc , Proteínas de Fluorescência Verde/análise , Humanos , Estrutura Terciária de Proteína , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Sequências Repetitivas de Aminoácidos , Deleção de SequênciaRESUMO
The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3'-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an alpha-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Delta5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of alpha-amanitin. Moreover, pol II harboring LS*Delta5 failed to transcribe up to the promoter-proximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.
Assuntos
RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Regiões 3' não Traduzidas/genética , Amanitinas/farmacologia , Animais , Linfoma de Burkitt , Clonagem Molecular , Genes fos , Proteínas de Choque Térmico HSP70/genética , Humanos , Substâncias Macromoleculares , Camundongos , Fosforilação , Regiões Promotoras Genéticas , RNA Polimerase II/química , Splicing de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Células Tumorais CultivadasRESUMO
The carboxy-terminal domain of the large subunit of mouse and human RNA polymerase II contains 52 repeats of a heptapeptide which are the targets for a variety of kinases. We have used an alpha-amanitin resistant form of the large subunit of pol II to study the role of the carboxy-terminal domain in the expression of chromosomal genes. The large subunit of RNA polymerase II and deletion mutants thereof, which contain only 31 (LSdelta31) and 5 (LSdeltaS) repeats, were expressed in 293 cells. Subsequently, the endogenous large subunit of RNA polymerase II was inhibited by alpha-amanitin and the induction of chromosomal c-fos and hsp70A genes was determined. Cells expressing the large subunit of RNA polymerase II and LSdelta31 were able to transcribe the c-fos and hsp70A genes after treatment with the phorbolester TPA and after heat-shock, respectively. In contrast, cells expressing LSdelta5 failed to induce expression of both genes.
