ABSTRACT
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , RNA Polymerase II , Transcription, Genetic , Transcriptional Elongation Factors , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/genetics , HSP70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , Positive Transcriptional Elongation Factor B/metabolism , Positive Transcriptional Elongation Factor B/genetics , Promoter Regions, Genetic , CRISPR-Cas Systems , Transcription Factors/metabolism , Transcription Factors/genetics , Polytene Chromosomes/genetics , Polytene Chromosomes/metabolism , Gene Expression Regulation , Phosphorylation , Protein Binding , Heat-Shock Response/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Nucleosomes/metabolism , Nucleosomes/geneticsABSTRACT
Atypical parkinsonism in Guadeloupe has been associated with the consumption of fruit and infusions or decoctions prepared from leaves of Annona muricata L. (Annonaceae), which contains annonaceous acetogenins, lipophilic inhibitors of complex I of the mitochondrial respiratory chain. We have determined the concentrations of annonacin, the major acetogenin in A. muricata, in extracts of fruit and leaves by matrix-assisted laser desorption-ionization mass spectrometry. An average fruit is estimated to contain about 15 mg of annonacin, a can of commercial nectar 36 mg, and a cup of infusion or decoction 140 microg. As an indication of its potential toxicity, an adult who consumes one fruit or can of nectar a day is estimated to ingest over 1 year the amount of annonacin that induced brain lesions in rats receiving purified annonacin by intravenous infusion.