Biomaterial Database

Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates. 1978

Y Groner, and E Gilboa, and H Aviv

HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm- capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008745	Methylation	Addition of methyl groups. In histo-chemistry methylation is used to esterify carboxyl groups and remove sulfate groups by treating tissue sections with hot methanol in the presence of hydrochloric acid. (From Stedman, 25th ed)	Methylations
D002467	Cell Nucleus	Within a eukaryotic cell, a membrane-limited body which contains chromosomes and one or more nucleoli (CELL NUCLEOLUS). The nuclear membrane consists of a double unit-type membrane which is perforated by a number of pores; the outermost membrane is continuous with the ENDOPLASMIC RETICULUM. A cell may contain more than one nucleus. (From Singleton & Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d ed)	Cell Nuclei,Nuclei, Cell,Nucleus, Cell
D006367	HeLa Cells	The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays.	Cell, HeLa,Cells, HeLa,HeLa Cell
D000546	Amanitins	Cyclic peptides extracted from carpophores of various mushroom species. They are potent inhibitors of RNA polymerases in most eukaryotic species, blocking the production of mRNA and protein synthesis. These peptides are important in the study of transcription. Alpha-amanitin is the main toxin from the species Amanitia phalloides, poisonous if ingested by humans or animals.	Amanitin
D012319	RNA Polymerase II	A DNA-dependent RNA polymerase present in bacterial, plant, and animal cells. It functions in the nucleoplasmic structure and transcribes DNA into RNA. It has different requirements for cations and salt than RNA polymerase I and is strongly inhibited by alpha-amanitin. EC 2.7.7.6.	DNA-Dependent RNA Polymerase II,RNA Pol II,RNA Polymerase B,DNA Dependent RNA Polymerase II
D012321	DNA-Directed RNA Polymerases	Enzymes that catalyze DNA template-directed extension of the 3'-end of an RNA strand one nucleotide at a time. They can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. (From Enzyme Nomenclature, 1992).	DNA-Dependent RNA Polymerases,RNA Polymerases,Transcriptases,DNA-Directed RNA Polymerase,RNA Polymerase,Transcriptase,DNA Dependent RNA Polymerases,DNA Directed RNA Polymerase,DNA Directed RNA Polymerases,Polymerase, DNA-Directed RNA,Polymerase, RNA,Polymerases, DNA-Dependent RNA,Polymerases, DNA-Directed RNA,Polymerases, RNA,RNA Polymerase, DNA-Directed,RNA Polymerases, DNA-Dependent,RNA Polymerases, DNA-Directed
D012359	tRNA Methyltransferases	Enzymes that catalyze the S-adenosyl-L-methionine-dependent methylation of ribonucleotide bases within a transfer RNA molecule. EC 2.1.1.	RNA Methylase,RNA Methylases,RNA, Transfer, Methyltransferases,T RNA Methyltransferases,tRNA Methyltransferase,Methylase, RNA,Methylases, RNA,Methyltransferase, tRNA,Methyltransferases, T RNA,Methyltransferases, tRNA,RNA Methyltransferases, T
D014158	Transcription, Genetic	The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.	Genetic Transcription