An RNA helicase (Dbp5) located on the cytoplasmic side of the NPC removes NXF1 and NXT1 from the mRNA in a reaction that is powered by ATP hydrolysis.

TLS Online TPP Program

#Id: 2642

#Unit 13. Methods in Biology

Notice that S1 mapping and primer extension are well suited to mapping transcripts made in vivo; by contrast, run-off transcription relies on transcription in vitro.

TLS Online TPP Program

#Id: 2643

#Unit 13. Methods in Biology

After you have identified the physiological transcription start site by S1 mapping or primer extension you can use run-off transcription in vitro.

TLS Online TPP Program

#Id: 2644

#Unit 13. Methods in Biology

A variation on the run-off theme of quantifying accurate transcription in vitro is the G-less cassette assay.

TLS Online TPP Program

#Id: 2645

#Unit 13. Methods in Biology

In G-less cassette transcription, a promoter is fused to a double stranded DNA cassette lacking G’s in the nontemplate strand, then the construct is transcribed in vitro in the absence of GTP. 

TLS Online TPP Program

#Id: 2646

#Unit 13. Methods in Biology

A G-less cassette, or stretch of nucleotides lacking guanines in the nontemplate strand, is inserted just downstream of the promoter. This template is transcribed in vitro with CTP, ATP, and UTP, one of which is labeled, but no GTP.

TLS Online TPP Program

#Id: 2647

#Unit 13. Methods in Biology

In G-less cassette, transcription will stop at the end of the cassette where the first G is required, yielding an aborted transcript of predictable size (based on the size of the G-less cassette, which is usually a few hundred base pairs long).