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

Question

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 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 polyadenylation signal for mRNA). The length of the various regions are indicated in the figure. a)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 size would it be, what structural features would it have, etc.? (5 pts) b)The Death Angel mushroom owes its awesome toxicity to the compound alpha-amanitin. It acts by inhibiting RNA polymerase. At what concentrations of alpha-amanitin would you expect transcription of the above construct to be inhibited, high, medium, low, or some combination? Why? (5 pts)

Explanation / Answer

Ans 1 :

(b) Alpha amanitin is produced by poisonous mushroom Amanita phalloides. This is also known as death cup. Lower concentrations (0.1- 1 µg/ml) of alpha amanitin strongly and selectively inhibits in vivo RNA synthesis. Alpha amanitin has a strong affinity to RNA polymerase II. Thus it inhibits transcription by binding to RNA polymerase II and it is unable to translocate once the DNA base is correctly base paired to RNA base in transcription.

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