Bc

DNA plus DNase I

Fragments missing (footprint)

Fragments missing (footprint)

The start site for transcription may be located by isolating mRNA and using reverse transcriptase to make complementary DNA.

Location of the Start of Transcription by Primer Extension

To fully understand the transcriptional regulation of a gene we need to know where transcription actually starts. Primer extension allows precise location of the start of transcription to the exact nucleotide. This approach involves binding of an artificial primer to messenger RNA. The primer, which is a DNA oligonucleotide, is then extended by reverse transcriptase, thus synthesizing DNA that is complementary to the mRNA (Fig. 25.13).

Since mRNA is needed, cells must first be grown under conditions where the gene of interest is highly expressed. The total mRNA is then extracted. An artificial DNA primer is synthesized so that it is complementary to a sequence close to the suspected start of transcription. The primer should be specific for the gene of interest and should therefore hybridize only with mRNA from this gene. The primer is hybridized to the mRNA and is extended by using reverse transcriptase.

The DNA/RNA hybrid is denatured and run on the same type of denaturing gel used in DNA sequencing (see Ch. 24). The extended DNA product must be labeled in some manner to allow visualization, either by radioactivity or fluorescence. The primer itself may be labeled or labeled nucleotides may be used during the extension step. The same primer as used for primer extension is also used to carry out a sequencing reaction using DNA corresponding to the transcribed region as template. The primer extension product will migrate to the same place size as the sequencing fragment that represents the precise transcriptional start site.

primer extension Method to locate the 5' start site of transcription by using reverse transcriptase to extend a primer bound to mRNA so locating the 5'-end of the transcript

Location of the Start of Transcription by S1 Nuclease 707

DNA Promoter 5'-UTR Coding sequence 3'-UTR Start of transcription

Transcription mRNA

1) EXTRACT RNA 2) Add DNA PRIMER

FIGURE 25.13 Primer Extension Reveals Start of Transcription

First, messenger RNA is isolated from cells that are expressing the gene of interest. A primer specific to the gene of interest is added and anneals to the mRNA. Reverse transcriptase makes a complementary DNA strand from the primer to the 5' end of the mRNA (i.e. the start of transcription). The length of the primer extension DNA strand is found by running it on a gel next to a sequencing ladder of the same region of DNA. This allows determination of the exact start site.

mRNA

DNA primer

Elongate with reverse transcriptase mRNA

DNA strand is elongated until it reaches start of transcription

Denature and run on sequencing gel

Was this article helpful?

0 0

Post a comment