Question 6 The following diagram represents the organization of a typical eukary

Question

Question 6

The following diagram represents the organization of a typical eukaryotic gene. (non-template strand shown)

The sequence AATAAA (AAUAAA in pre-mRNA) is the Poly(A) signal sequence recognized by CPSF (cleavage and polyadenylation specificity factor).

1) Nuclear and cytoplasmic fractions were purified from eukaryotic cells and RNA was purified from each fraction. A careless student labeled the RNA samples, #1 and #2, but forgot which one was extracted from the cytoplasmic fraction. To determine the origin of samples #1 and #2, you decide to perform the following experiment:

Each sample is submitted to gel electrophoresis to resolve RNA molecules based on their size. After electrophoresis, the RNA content of the gel is transferred onto a membrane. The membrane is probed with a radiolabeled oligonucleotides: ACAACCACAC and then placed into contact with an X-ray film to determine the position of the RNA molecules recognized by the radioactive probe.
A similar experiment is conducted using a different radioactive probe whose sequence is TTTATT.

The developed X-ray films are shown in the following figure.

a) During polyadenylation, what is the function of the pre-mRNA sequence corresponding to “GTGTGGTTGT” on the non-template DNA strand?

b) Identify the sample (#1 or #2) extracted from the cytoplasmic fraction. Give two reasons to explain your answer.

Explanation / Answer

a) the pre-mRNA sequence corresponding to “GTGTGGTTGT” on the non-template DNA strand is telomeric DNA sequence which is recognized by telomerase enzyme. only pre-mRNA will have that sequence, mature mRNA do not have this sequence.

b) sample 1 is extracted from the cytoplasm and 2 from nucleus.

1) sample 2 binds with telomeric region-specific probe and poly A specific probe. while sample 1 is bound with poly A specific probe only.

2) the Mw of sample 2 is more as compared to the sample 1. in nucleus pre-mRNA is converted into mature mRNA . so intron is removed and unnecessary sequence is removed. due to this mature mRNA is shorter than pre-mRNA molecule.

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