Nucleic Acids Research, Vol 26, Issue 4 919-924, Copyright © 1998 by Oxford University Press
A Law, K Hirayoshi, T O'Brien and JT Lis
A new method is described for cloning DNA sequences occupied by a specific
protein on chromatin in vivo . The approach uses UV cross- linking to
couple proteins covalently to DNA and the resulting complexes are then
purified under stringent conditions. Particular adducts are
immunoprocipitated with antibody to the protein of interest. The resulting
DNA (iDNA) is amplified by PCR, cloned and characterized. The model system
used was RNA polymerase II (Pol II), whose density on particular DNAs under
various conditions is well documented. Pol II can exist in several states
on DNA. While Pol II can simply be bound to DNA, the bulk of DNA-associated
Pol II is transcriptionally engaged in either the transcribing or paused
states. Paused Pol IIs that have previously been characterized are found at
promoters and have the distinctive property that their transcription in
isolated nuclei is stimulated by sarkosyl or high salt. Here we isolate and
sequence DNAs that cross-link to Pol II molecules. We identify by nuclear
run-on assays those DNAs that have Pol II engaged in transcription. Twenty
one percent of the iDNA clones that have detectable transcriptionally
engaged Pol II appear to be paused, in that they display
sarkosyl-stimulated trancription in a nuclear run-on transcription assay.
At least some of these map to the 5'-ends of genes. These results suggest
that transcriptional pausing of Pol II is a general phenomenon in vivo.
ARTICLES
Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila
Section of Biochemsitry, Molecular and Cellular Biology, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.
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