Open AccessTranscription of Ribosomal 5‐S RNA by RNA Polymerase C in Isolated Chromatin from HeLa Cells
Author(s) -
YAMAMOTO Mikio,
JONAS Dagmar,
SEIFART Klaus
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11876.x
Subject(s) - rna , microbiology and biotechnology , rna polymerase i , rna dependent rna polymerase , transcription (linguistics) , biology , rna polymerase , small nuclear rna , polymerase , nuclease protection assay , chromatin , dna , rna polymerase ii , rna polymerase iii , ribosomal rna , biochemistry , gene expression , gene , promoter , linguistics , philosophy
The synthesis of ribosomal 5‐S RNA in vitro in isolated chromatin from HeLa cells was studied by hybridization of the product to a plasmid DNA probe which contained the 5‐S RNA genes from Xenopus mulleri . It was found that endogenous RNA polymerase C, contained in the chromatin, actively synthesizes RNA which hybridizes to the DNA for 5‐S RNA. The molecular weight of this RNA, judged by gel filtration and electrophoresis in formamide‐containing polyacrylamide gels, is of distinct 5‐S size. Hybridisation of the product synthesized in vitro to the DNA for 5‐S RNA can be competed quantitatively by the presence of unlabelled cytoplasmic 5‐S RNA. Presaturation of the DNA‐containing filters with cytoplasmic 5‐S RNA prevents hybridisation, indicating that antisense transcripts are not synthesized to a measurable extent in this system. The addition of Escherichia coli RNA polymerase to chromatin stimulates the synthesis of bulk RNA, and beyond certain concentrations also leads to enhanced formation of hybridizable sequences. These are, however, predominantly contained in molecules of high molecular weight, indicating abberent initiations and/or terminations of the bacterial polymerase at random sites. These data re‐emphasize the necessity of concurrent hybridization and size analyses for the assay of specific transcription. Homologous RNA polymerase C, added to chromatin either as a crude preparation in the presence of polymerase A or as purified enzyme, resulted in increased production of hybridizable 5‐S RNA sequences, the formation of which was suppressible by high concentrations of α‐aminitin. The RNA products synthesized under these conditions were predominantly asymmetric and hybridized in a discrete peak at the 5‐S position. This is in contrast to results found for the bacterial enzyme which synthesized a high proportion of antisense transcripts on isolated chromatin. These findings indicate strongly that a particular RNA polymerase species of homologous source should be used when analyzing the transcription of specific genes in vitro .