Question

A 3.1-kb fragment of DNA containing a complete human gene has been made available to you. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site for the gene. You decide to do an electromobility shift assay (EMSA) to determine what molecules bind to the fragment. The results are shown below. If Lane 1 contains only the 3.1-kb fragment, which answer correctly describes another lane of the experiment?

1 2 3

a. Lane 2 contains the 3.1kb fragment + TFIIB

b. Lane 2 contains the 3.1kb fragment + RNA polymerase II

c. Lane 2 contains the 3.1kb fragment + TFIIA

d. Lane 2 contains the 3.1kb fragment + TFIID

e. Lane 3 contains the 3.1kb fragment + TFIIB + RNA polymerase II

Answer 1

During transcription initiation the TATA box promoter region will be recognized by TATA binding protein (TBP) portion of transcription factor D (TFIID), which further leads to the formation of transcription complex, recruiting other transcription factors like TFIIA, TFIIB, TFIIF, TFIIH. To study DNA and protein interactions, electromobility shift assay (EMSA) is used to detect such interactions which if present will make DNA molecule heavier and thus slow moving during electrophoresis. Therefore when DNA fragment 3.1kb fragment is associated with transcription factor TFIIB along with RNA polymerase II, then its mobility will be slower as compared to bands which are either only 3.1kb DNA or associated with transcription factors like TFIIB, TFIIA, TFIID or with RNA polymerase II.

Therefore, correct option is e. Lane 3 contains the 3.1kb fragment + TFIIB + RNA polymerase II