Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II
Eukaryotic RNA polymerase II consists of three subspecies, designated II/sub 0/, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with ..gamma..(/sup 32/P)-ATP in the presence of casein kinase I and /sup 32/P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of /sup 32/P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription.
- Research Organization:
- Univ. of California, Davis
- OSTI ID:
- 5019596
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CHROMATOGRAPHY
CLEAVAGE
CRYSTAL STRUCTURE
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
GENES
IMMUNE REACTIONS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MICROSTRUCTURE
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
POLYMERASES
PROTEINS
PURIFICATION
RADIOISOTOPES
RNA POLYMERASES
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES