Figure 7.

Mapping the Pol II Transcription Start Site on Type 3 Pol III Promoters

(A) Schematic of the mRNA 5′-RACE procedure. The Cap-Clip Acid Pyrophosphatase is used to specifically hydrolyze the 5′ cap of mRNA. (B) Amplification of Pol II-specific transcripts made by the three promoters. The 5′-RACE was performed with (+) or without (−) Cap-Clip treatment. Products of the expected size are marked by a black triangle. (C) Illustration of Pol II start site usage on Pol III promoters. The Cap-Clip-dependent products from (B) were subjected to TA-cloning and Sanger sequencing. The Pol II transcription start site was determined by aligning the sequencing output with the DNA construct. The start position was related to the position of Pol III transcription initiation, which was arbitrarily defined as +1. (D) Sequences around the Pol III/II transcription start sites. Pol III start sites (solid arrow) and Pol II start sites (dotted arrow) are indicated. The TATA boxes of these Pol III promoters are underlined and the position is indicated. The relative Pol III and II strengths (right panel) were derived from the results represented in Figure 1.