Mercurated nucleotides: assessment of a new tool to study RNA synthesis and processing in isolated nuclei.
Mercurated pyrimidine nucleotides have been used to study RNA synthesis and processing in isolated nuclei from mouse L cells. 5-mercuridine triphosphate (5-Hg-UTP) or 5-Hg-CTP are accepted as substrates by the purified RNA polymerases (I+III) and (II) from mouse cells, respectively, as well as by the enzymes still bound to the nuclear chromatin. In nuclei, RNA synthesis in the presence of Hg-UTP is reduced to 60-70% of a control. 30-60% of RNA labeled in vitro with (3H)UTP in isolated nuclei is not retained on sulfhydryl sepharose columns. Sucrose gradient analysis reveals a size distribution of the non-bound RNA similar to non-mercurated control RNA. Hg-RNA is found in a single peak from 4-10S. Chase experiments indicate that this RNA is the original transcript. It is argued that Hg-nucleotides may cause premature chain termination. Methylation of RNA in vitro by S-adenosyl methionine ((3H)SAM) is reduced to 75% of controls in the presence of Hg-UTP. Only 6% of the methyl groups appear in Hg-RNA. Polyadenylation is reduced as well. 15% of poly(A) (+)RNA are found in control assays whereas only 1% of Hg-RNA carries a poly(A) end added in vitro. These results limit the use of mercurated nucleotides for studies of nuclear RNA synthesis and processing.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D008722:Methods; D009941:Organomercury Compounds; D012313:RNA / Q000096:biosynthesis* / Q000302:isolation & purification; D012265:Ribonucleotides
D009941:Organomercury Compounds; D012265:Ribonucleotides; D012313:RNA; D012321:DNA-Directed RNA Polymerases