You have isolated different recombinant cDNA clones from pancreas mRNA. Based on

Question

You have isolated different recombinant cDNA clones from pancreas mRNA. Based on their DNA sequence, you suspect the cDNA clones encode a protein that binds to the promoter of an insulin receptor gene and results in transcriptional activation (Figure 2A). The clones extend for different distances toward the 5’ end of the gene. The clones were transcribed and translated in vitro, and the translated proteins were mixed with 32P-labeled DNA containing the recognition sequence for the activator protein. Either radioactive DNA without added protein (lane 1), or mixtures containing DNA and different segments of the activator protein were subjected to DNA mobility shift assays (Figure 2B). Lanes 2-5 contain protein synthesized from clones 1-4 incubated with 32P-labeled DNA, respectively, and lane 6 contains protein from clones 3 and 4 incubated with 32P-labeled DNA. Protein/32P-labeled DNA complexes were separated by polyacrylamide gel electrophoresis and auto-radiographed to visualize the labeled DNA fragments.  The intensity of the labeled DNA bands reflects their concentration. The gel slot shows the positions where the samples were loaded; electrophoresis occurs from top (anode) to bottom (cathode). Hint: Protein size and multimeric state can influence migration of protein/DNA complexes.

1. Why does clone 1 fail to bind DNA?

2. There are three shifted bands when clones 3 and 4 are mixed together. What does that tell you about the structure of the gene regulatory protein?

Figure 2. (A) MAP OF THE CLONE 2 4 5' (B) GEL-MOBILITY SHIFT ASSAY 2 1 2 3 4 5 6 Figure 2. Gene segments encoding transcription activator (A) were transcribed and translated in vitro. The resulting proteins were used in gel mobility shift assays to identify those that can bind their cognate DNA binding site.

Explanation / Answer

1. Clone 1 does not contain most of the 5'-region of the gene. This suggests that the DNA binding domain of the protein is present at the 5'-end of the gene. hence clone 1 did not bind to the probe.

2. When two different proteins (varying in lengths) are mixed and incubated with the probe, three bands were observed. This suggests that the protein can form higher order structures such as dimeric and trimeric forms.

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