The sequence AAUAAA (hexamer) is a signal for cleavage to generate a 3’ end of mRNA that is polyadenylated.

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#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.

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#Unit 13. Methods in Biology

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

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#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. 

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#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.

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#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).

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#Unit 13. Methods in Biology

Primer extension, S1 mapping, and Northern blotting are useful for determining the concentrations of specific transcripts in a cell at a given time, but they do not necessarily tell us the rates of synthesis of the transcripts.