ABSTRACT
Analysis of the distribution of RNA polymerase II on the genomes of Drosophila and human cells using in vivo protein-DNA crosslinking reveals that RNA polymerase II (Pol II) is concentrated at the 5'-ends of thousands of genes. This appears to be irrespective of transcription levels. Hence, a potential regulatory step in the transcription of many genes occurs after Pol II has associated with the promoter. The protein-DNA crosslinking technique widely used to monitor Pol II and other proteins on chromosomes in vivo, however, does not reveal if Pol II is transcriptionally engaged on DNA. Genomic footprinting with potassium permanganate provides one method for detecting transcriptionally engaged Pol II. Using this approach, we have determined that the Pol II associated with the promoters of many genes has initiated transcription but paused in the region 20-50 nucleotides from the start. Here we describe the application of this method in Drosophila and human cells. The method should prove useful in assessing if promoter bound Pol II has engaged in transcription and for investigating the establishment and regulation of transcriptionally engaged Pol II.
Subject(s)
DNA Footprinting , DNA-Directed RNA Polymerases/physiology , Manganese Compounds , Oxides , Transcription, Genetic , Animals , Base Sequence , Drosophila , Humans , Molecular Biology/methods , Molecular Sequence Data , Polymerase Chain ReactionSubject(s)
Gene Expression Regulation , Transcription, Genetic , DNA-Binding Proteins/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genome , Operon/genetics , Peptide Elongation Factors/geneticsABSTRACT
NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and beta1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.
