With the advent of recombinant DNA technology ?t became possible to construct hy

Question

With the advent of recombinant DNA technology ?t became possible to construct hybrid genes with sequences from different sources fused together. Perhaps surprisingly, DNA sequences such as promotors or . f . , , . 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 major rRNA genes (normally transcribed by RNA polymerase I) with the coding sequence of a tRNA gene (normally transcribed by RNA polymerase III). In addition, just upstream of the termination site of the tRNA a 3?- AAAAAAT-5? sequence was inserted (this is the polyad?nylation signal for mRNA). The length of the various regions are indicated in the figure. Site of initiation . p p i Site of termination I ? ? ? I e ? .... La.... . . DNA from a tRNA gene . . - . - f__ I_he p ? ? ? ? ? ? ? Termination signal from a mRNA gene Promotor from rRNA gene This hybrid gene was transcribed in an ?n 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 size would it be, what structural features would it have, etc.? (5 pts) I The Death Angel mushroom owes its awesome toxicity to the compound a-amanitin. It acts by inhibiting RNA polymerase. At what concentrations of ct-amanitin would you expect transcription of the above construct to be inhibited, high, medium, low, or some c?mbination? Why? (5 pts) loo bp t r t 25bp I

Explanation / Answer

Based on the given data,

a)

The attached promoter is from rRNA gene, hence it is only recognized by the RNA pol-I. So, RNA pol-I binds on its promotor, no matter what is the remaining sequence the gene has it transcribes until it reach to its terminator sequence. The usual termination sequence of all RNA pols is Poly-A. Hence, when it reaches to Poly-A, it stalls away from the gene.

Thus, it only transcribes 100 base pairs (bp) of tRNA. However, the system also contains RNA Pol-II and Pol-III, but they cannot transcribe the provided gene, because they do not have a promotor sequence for binding.   

b)

The lethal dose of alpha-Amanitin is 0.1 mg/kg; it has very low range LD5O. Hence, if the LD50 value is low, then the chemical has more toxicity, and causes acute toxicity. For example, an organism has Wight 11 grams, then

0.1 × 11 = 1.1 mg of alpha-Amanitin would be enough to kill that organism. Hence, very low concentration of alpha-Amanitin can inhibit the transcription by inhibiting all RNA polymerases.   

   

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