With the advent of recombinant DNA technology it became poss

With the advent of recombinant DNA technology, it became possible to construct hybrid genes with sequences from different sources fused together. Perhaps surprisingly, DNA sequences such as promotors or terminators are autonomous, that Is, no matter what sequences they are connected to they retain their normal function. With this in mind, consider the following construct that fuses the promotor from the rRNA gene with the coding sequence of the 5S RNA gene. In addition, just upstream of the normal termination site of the 5S RNA, a 3-AAAAAAT-5\' sequence was inserted (this Is the polyadenylation signal for mRNA). The length of the various regions is indicated in the figure. This hybrid gene was transcribed in an in vitro transcription mixture including all three RNA polymerases and all the necessary ingredients for each to function properly. Predict the structure of the resulting transcript (or transcripts), i.e. what sue would it be, what structural features would It have, etc.? The Death Angel mushroom owes its awesome toxicity to the compound alpha-amanitin. it arts by inhibiting RNA polymerase. At what concentrations of alpha-amanitin would your expert transcription of the above construct to be inhibited, high, medium, low, or some combination? Why?

Solution

Please find the answers below:

Part a: According to the information, the gene encoding for 5S unit has been inserted in the linear structure of artificially constructed promoter region. Since the promoter region is autonomous in nature, it will definitely encode for the appropriate polypeptide chain which will generate the 5S protein. According to the data, it can be seen that the gene for 5S sequence is 100bp in length. We know that triplets of nucleotide make a codon and each codon encode for an amino acid. Thus, from this 100bp sequence, at least 100/3 or 33 amino acids can be synthesized. Thus, the length of amino acid sequence synthesized from transcription of this gene segment will be 33 amino acid long.

Secondarily, it is important to note here that the house-keeping genes are mostly comprised of essential amino acids which can be synthesized de nove from breakdown of other biomolecules in the body or are utmost required. Thus, the amino acids will be comprised of maximum possible essential amino acids. Structurally, this polypeptide must be linear in structure and attain its quarternary structure after post-transcriptional modifications.

Part b: The extract of alpha amanitin is highly toxic to cells. This poison kills the cells by inhibiting the binding of RNA polymerase to the promoter region. Even very small concentrations of this toxin are deleterious to cells. Thus, it can be expected that alpha amanitin would prevent transcription of this gene construct at even very low concentrations.