MCAT Biochemistry: Transcription Factors
A research team is studying the effects of a newly identified transcription factor, TF-X, on the expression of a gene that encodes an enzyme involved in nucleotide biosynthesis. To investigate, they transfect human cell lines with either an empty vector (control group) or a plasmid encoding TF-X (experimental group). After 24 hours, they measure mRNA levels of the target gene using quantitative PCR (qPCR). They observe that mRNA levels are significantly higher in the experimental group compared to the control. To confirm that TF-X binds directly to the promoter of the target gene, they perform a chromatin immunoprecipitation (ChIP) assay using antibodies specific to TF-X. The ChIP data indicate enriched binding of TF-X to a region of DNA upstream of the target gene.
Which of the following control experiments would best validate that TF-X's effect on target gene expression is transcriptionally mediated?
A) Perform a Western blot to detect the protein product of the target gene in both control and experimental groups.
B) Use a plasmid encoding a TF-X mutant lacking the DNA- binding domain and measure the mRNA levels of the target gene.
C) Knockdown TF-X expression using siRNA and measure the protein product of the target gene using an ELISA assay.
D) Perform an electrophoretic mobility shift assay (EMSA) using the TF-X protein and a labeled DNA fragment containing the target gene promoter region.
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