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Translational (Protein) Fusion

Translational Gene Fusion is used to measure protein localization by quantifying promoter activity.

The fusion results in a gene with a promoter, fluorescent protein gene (usually yellow fluorescent protein YFP or green fluorescent protein GFP), the gene of interest itself and a transcriptional stop codon. There is no stop codon between GFP and the gene of interest, and thus a fusion protein is encoded: our protein of interest is tagged with GFP.

This type of construct allows you to locate the protein of interest within the cell, and detect changes in σ protein stability or activity.

In an experiment where we look at promoter activity we use a GFP to mesaure promoter and protein activity of sigma (σ) factor.

First the GFP gene is introduced onto the σ promoter. Then transcription produces a single mRNA with two fused genes: σ and GFP. Then since there is no stop codon between σ and GFP, translation gives a single polypeptide comprised of 2 fused proteins: σ fused to GFP. The stop codon is placed in the mRNA at the end of the σ gene.

Remember that experimental controls do two things: rule out alternative explanations for results of experiment; and verify that the experimental set-up works and why. A common experimental control is to measure untagged cells for GFP activity, and to cross-check by assaying for mRNA.

Translational (Protein) FusionComments