Product Pathways - Chromatin Regulation / Epigenetics
Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb #13523
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Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, ChIP=Chromatin IP, ChIP-seq=Chromatin IP-seq,
Species predicted to react based on 100% sequence homology: Hamster, D. melanogaster, Xenopus, Bovine, Pig, S. cerevisiae, C. elegans,
Specificity / Sensitivity
Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb recognizes endogenous levels of Rpb1 only when the carboxy-terminal domain (CTD) heptapeptide repeat [Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7] is phosphorylated at Ser5. This antibody does not cross-react with Rpb1 CTD phosphorylated at Ser2 or Ser7.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser5 of the human Rpb1 CTD heptapeptide repeat.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human β-Actin Promoter Primers #13653, human β-Actin intron 1 primers, SimpleChIP® Human β-Actin 3' UTR Primers #13669, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Western blot analysis of extracts from C2C12, H-4-II-E, and COS-7 cells using Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb.
Peptide dot blot analysis demonstrating Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb antibody specificity. Antibody binding to pre-coated Rpb1 CTD peptides is shown using Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499, Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb, a phospho-Rpb1 CTD (Ser7) antibody, and Phospho-Rpb1 CTD (Ser2/Ser5) (D1G3K) Rabbit mAb #13546. As expected, the Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb only binds to phospho-Rpb1 CTD peptide when phosphorylated at Ser5.
Immunoprecipitation of Rpb1 from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 Hela cells and either 10 μl of Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb or 10 μl of Rpb1 NTD (D8L4Y) Rabbit mAb #14958, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 5ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across the ZNF740 gene on chromosome 12. For additional ChIP-seq tracks, please download the product data sheet.
RNA polymerase II (RNAPII) is a large multi-protein complex that functions as a DNA-dependent RNA polymerase, catalyzing the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates (1). The largest subunit, RNAPII subunit B1 (Rpb1), also known as RNAPII subunit A (POLR2A), contains a unique heptapeptide sequence (Tyr1,Ser2,Pro3,Thr4,Ser5,Pro6,Ser7), which is repeated up to 52 times in the carboxy-terminal domain (CTD) of the protein (1). This CTD heptapeptide repeat is subject to multiple post-translational modifications, which dictate the functional state of the polymerase complex. Phosphorylation of the CTD during the active transcription cycle integrates transcription with chromatin remodeling and nascent RNA processing by regulating the recruitment of chromatin modifying enzymes and RNA processing proteins to the transcribed gene (1). During transcription initiation, RNAPII contains a hypophosphorylated CTD and is recruited to gene promoters through interactions with DNA-bound transcription factors and the Mediator complex (1). The escape of RNAPII from gene promoters requires phosphorylation at Ser5 by CDK7, the catalytic subunit of transcription factor IIH (TFIIH) (2). Phosphorylation at Ser5 mediates the recruitment of RNA capping enzymes, in addition to histone H3 Lys4 methyltransferases, which function to regulate transcription initiation and chromatin structure (3,4). After promoter escape, RNAPII proceeds down the gene to an intrinsic pause site, where it is halted by the negative elongation factors NELF and DSIF (5). At this point, RNAPII is unstable and frequently aborts transcription and dissociates from the gene. Productive transcription elongation requires phosphorylation at Ser2 by CDK9, the catalytic subunit of the positive transcription elongation factor P-TEFb (6). Phosphorylation at Ser2 creates a stable transcription elongation complex and facilitates recruitment of RNA splicing and polyadenylation factors, in addition to histone H3 Lys36 methyltransferases, which function to promote elongation-compatible chromatin (7,8). Ser2/Ser5-phosphorylated RNAPII then transcribes the entire length of the gene to the 3' end, where transcription is terminated. RNAPII dissociates from the DNA and is recycled to the hypophosphorylated form by various CTD phosphatases (1).
In addition to Ser2/Ser5 phosphorylation, Ser7 of the CTD heptapeptide repeat is also phosphorylated during the active transcription cycle. Phosphorylation at Ser7 is required for efficient transcription of small nuclear (sn) RNA genes (9,10). snRNA genes, which are neither spliced nor poly-adenylated, are structurally different from protein-coding genes. Instead of a poly(A) signal found in protein-coding RNAs, snRNAs contain a conserved 3'-box RNA processing element, which is recognized by the Integrator snRNA 3' end processing complex (11,12). Phosphorylation at Ser7 by CDK7 during the early stages of transcription facilitates recruitment of RPAP2, which dephosphorylates Ser5, creating a dual Ser2/Ser7 phosphorylation mark that facilitates recruitment of the Integrator complex and efficient processing of nascent snRNA transcripts (13-15).
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