In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.
The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012315:RNA Caps / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D006360:Heat-Shock Proteins; D012315:RNA Caps; D012321:DNA-Directed RNA Polymerases