The diagram below shows the DNA sequence of a gene which is expressed only in th
ID: 66986 • Letter: T
Question
The diagram below shows the DNA sequence of a gene which is expressed only in the skin of the Jackalope, a diploid eukaryote. Shown is DNA sequence from close to the transcriptional start site, as well as some upstream sequence. The first nucleotide transcribed is an A, and the corresponding A on the coding strand is underlined. Two other sequences in this region are underlined.
Below the sequence is shown the structure of several pieces of DNA that I tested for their ability to initiate transcription. Construct a is the wild-type sequence and b through f either contain small deletions (b-d) or point mutations (e, f). At the right are indicated the relative levels of transcription for each construct, in Arbitrary Units.
i. For each mutant construct (b-f), indicate: what sequence element might be affected and why the expression level is changed to the extent shown.
I was also able to isolate an enhancer for this gene, which could further increase the levels of expression of the promoter in Jackalope skin cells. The sequence of the enhancer is shown below.
My research has also shown that two transcription factors acting upon this gene are Mattigen and Tammigen. These proteins bind to DNA at the sequences 5’AATT3’ (for Mattigen) and 5’CCCGGG3’ (for Tammigen).
Below the sequence are the structures of five constructs: a wild-type enhancer (g), and four deletion mutants (h through k). These constructs were tested for their ability to help the promoter to increase the level of transcription. Relative levels of expression are shown, once again measured in Arbitrary Units.
ii. For each mutant construct (h-k), indicate which transcription factor’s function might be affected, and suggest why the expression level is changed to the extent shown
Explanation / Answer
i
b. The GGGCGG site is a proximal binding element present upstream of the TATA box in eukaryotes. This sequence regulates the true transcription of DNA. When this sequence is deleted, only basal level of transcription takes place.
c. The sequence has TATA box deleted, a place where RNA polymerase binds. This affects the overall level of the transcription.
d. In this sequence both proximal binding element and TATA box are deleted, due to which the gene cannot transcribe at all.
e. The point mutation T instead of A is present in promoter binding sequence. It has no effect on the transcription because the RNA polymerase recognises the first four sequences TATA for initiating the transcription. That is why there is an increase in the activity compared to the wildtype.
f. The point mutation G is present in the TATA region of promoter binding which affects the initiation of the transcription. This is the reason decrease in the relative activity.
h. Deletion of one of the transcription factors binding site (Mattigen) has decreased the activity by half, which suggests that both the TF binding is necessary for full activity.
i. Deletion of another TF binding site (Tammigen) has decreased the activity by half.
j. Deletion of both TF binding sites has an additive effect decreasing the activity by 10 folds.
k. The CCCGGG is a kozak consensus sequence which is important for the initiation of translation in eukaryotes. The deletion of this sequence has the same effect as deleting the TF binding sites.