Question 3: Scientists have introduced in bacteria a wild type gene coding for G

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Question 3:

Scientists have introduced in bacteria a wild type gene coding for GFP (green fluorescent protein) or two engineered versions of this gene containing an additional cis-regulatory element (crRL or crR12). The transcription of the wild-type GFP gene leads to the production of an mRNA containing a cis-acting element labeled X. Transcription of the engineered GFP genes leads to the production of mRNAs containing the cis- acting X and either crRL or crR12 in the 5’ portion of the mRNA. The synthesized mRNA molecules are diagrammed below.

1) To identify the function of X, scientists performed the following assay: purified small ribosomal subunits, large ribosomal subunits, 16S rRNA (ribosomal RNA), or Alanyl-tRNA were bound to a filter and the filter was then incubated with radioactive wild type GFP-mRNA molecules (with or without the cis-acting element X). The radioactivity associated with the filter was measured after removal of the unbound radioactive material. The results are given in the following table.

Based on these data and your knowledge of translation in prokarytotes determine the function of X. Briefly explain.

Wild-type GFP mRNA GFP (Open reading frame) 5 AUG STOP3' GFP-mRNA Engineered GFP mRNA CrRL or CrR12 X GFP (Open reading frame) CrRL mRNA or STOP3' crR12mRNA

Explanation / Answer

In prolaryotic translation, there is a purine rich region in the prokaryotic mRNA called the Shine- Dalgarno (SD) sequence, which is complementary to a pyrimidine rich sequence in the 16SrRNA of the 30S ribosomal subunit.Complementary pairing of these two RNAs will hold the mRNA to the 30S subunit to initiate the transcription by placing the start codon(AUG) to the psite of the ribosome.

from the experimental date it is clear that the radioactive wild typeGFP mRNA has been bound to 16SrRNA(2300 counts per minute) as well as 30S subunit(1500 counts per minute) and not to the large subunit or the Ala- tRNA.

So it can be concluded that X works as the SD sequence

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