Limited accessibility of chromatin satellite DNA to RNA polymerase from Escherichia coli.
An attempt was made to elucidate some of the factors influencing the fidelity with which isolated chromatin from mouse L-cells is transcribed by RNA polymerase from Escherichia coli by analyzing the in vitro transcript for the presence of satellite sequences. These sequences are absent from cellular RNA and therefore reflect aberrant transcription. The results indicate that satellite sequences are underrepresented in chromatin transcripts relative to those of DNA. This selectivity is insensitive to many variables in procedures for the isolation and transcription of chromatin. However, lowering the ratio of enzyme to template further reduced the proportion of satellite sequences in the transcript. We conclude that a primary factor influencing the extent of aberrant transcription is the level of enzyme used. Under limiting enzyme conditions, an efficient selection against satellite sequences is observed. However, under conditions of enzyme excess, the enzyme initiates chains at weaker secondary promoters localized in regions of the chromatin containing satellite DNA.
D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D009693:Nucleic Acid Hybridization; D014158:Transcription, Genetic
D002843:Chromatin; D004247:DNA; D012321:DNA-Directed RNA Polymerases