Product Usage Information
For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 10 6 cells) per IP. This antibody has been validated using SimpleChIP ® Enzymatic Chromatin IP Kits. The CUT&RUN dilution was determined using CUT&RUN Assay Kit #CUT&RUN Assay Kit # 86652 .
Western Blotting: 1:1000
Chromatin IP: 1:50
CUT&RUN: 1:50
Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at -20°C. Do not aliquot the antibody.
Protocol
Available protocols: Western Blotting, Chromatin IP, CUT&RUN
Specificity / Sensitivity
Phospho-Rpb1 CTD (Thr4) (D7L9W) Rabbit Monoclonal Antibody #26319 recognizes endogenous levels of Rpb1 only when the carboxy-terminal domain (CTD) heptapeptide repeat [Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7] is phosphorylated at Thr4. This antibody does not cross-react with Rpb1 CTD phosphorylated Ser2, Ser5, or Ser7.
Species Reactivity: Human, Mouse, Rat, Monkey
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr4 of human Rpb1 CTD heptapeptide repeat.
Background
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). Phosphorylation of the Rpb1 CTD heptapeptide repeat at Thr4 is highly conserved from yeast to mammals. However, research studies using Thr4 phosphorylation-mutant Rpb1 proteins suggest different roles for this modification among species. While phosphorylation of Thr4 in yeast is not essential (16,17), Thr4 mutants in chicken and mammalian systems result in RNA processing errors and global defects in RNA elongation (18,19). Threonine 4 is directly phosphorylated by polo-kinase3 (PLK3) and cyclin dependent kinase-9 (CDK9) activity is thought to either directly or indirectly lead to the phosphorylation of this site (18,19).
Alternate Names
DNA-directed RNA polymerase II largest subunit, RNA polymerase II 220 kd subunit; DNA-directed RNA polymerase II subunit A; DNA-directed RNA polymerase II subunit RPB1; DNA-directed RNA polymerase III largest subunit; hRPB220; hsRPB1; MGC75453; NEDHIB; POL II; POLR2; POLR2A; POLRA; polymerase (RNA) II (DNA directed) polypeptide A, 220kDa; polymerase (RNA) II subunit A; RNA polymerase II subunit A; RNA polymerase II subunit B1; RNA-directed RNA polymerase II subunit RPB1; RNAPII; RPB1; RPBh1; RpIILS; RPO2; RPOL2
