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Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III
Author(s) -
Pombo Ana,
Jackson Dean A.,
Hollinshead Michael,
Wang Zhengxin,
Roeder Robert G.,
Cook Peter R.
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.8.2241
Subject(s) - biology , polymerase , rna polymerase i , rna polymerase ii , transcription factories , transcription (linguistics) , rna polymerase iii , nucleoplasm , microbiology and biotechnology , nucleolus , rna polymerase , rna polymerase ii holoenzyme , transcription factor ii d , rna , rna dependent rna polymerase , biochemistry , gene expression , dna , gene , promoter , nucleus , linguistics , philosophy
Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved ‘physiological’ buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br‐UTP, before the resulting Br‐RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that ∼10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only ∼2000 sites (diameter ∼40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or ‘factories’, suggesting that different regions of the nucleus specialize in the transcription of different types of gene.