Figure 5

Analyses of degradation processes on labeled transcript accumulation by pulse-chase labeling in run-on (A, C) and RePro systems (B, D). After 20-min transcription in the presence of [³²P]-UTP (t₀), unlabelled UTP was added to the reaction mixture (+UTP), and the reaction was let run further for 15 (t₁₅) or 30 min (t₃₀). Control reactions without addition of unlabeled UTP were performed in parallel (-UTP). (A) Dot-blot analysis of a typical run-on reaction hybridized to rrn16 DNA fragments spotted in triplicates. (B) 5'-ends of rrn16 transcripts were analyzed by RNase Protection Assay (RPA, lane 2) and Reverse RNase Protection Assay at indicated points of time with or without the addition of unlabeled UTP (lanes 3-7). A control for self-protection of labeled rrn16 transcripts after 35 min of reaction time, however, without the addition of unlabeled asRNA is shown in lane 8. Mapped rrn16 transcription initiation site (filled circle) is identified by its distance between the transcription initiation site and the first nucleotide of the mature rRNA. Molecular weight marker in nt is provided on the side (M, lane 1). An additional signal of about 130 bp of an potentially elongation arrested transcript is marked by an asterisk. Data shown for the representative autoradiograms may slightly differ from the averages of multiple experiments shown in (C and D). (C) Relative rates of transcription (× 1000) for the rrn16 gene as determined by run-on analyses in (A). (D) Relative rates of transcription (× 1000) for the rrn16 gene as determined by RePro in (B).