With the advent of recombinant DNA technology it became possible to construct hy
ID: 35955 • Letter: W
Question
With the advent of recombinant DNA technology it became possible to construct hybrid genes with seqences from different sources fused together. Perhaps surprisely, DNA sequences such as promotors or terminators are autoonomous, that is, no matter what sequences they are connected to they retain theri normal function. With this in mind, consider the following construct that fuses the promotor from the major rRNA genes (normaly 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 ofthe various regions are indicated in the figure:
Draw a diagram for A and B
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 transcipt (or transcripts), i.e., what size would it be, what structural features would it have, etc?
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.Explanation / Answer
The small segment of RNA, which is a DNA dependent RNA polymerase promotes initiation by acting as the primer site for the DNA polymerase.
and ofcourse the promoters-Modern multipurpose cloning vectors contain a multiple cloning site (MCS) flanked on each side by promoters for different polymerases. This allows the synthesis of either sense or antisense RNAs from sequences cloned into the multiple cloning site. In addition to plasmid DNA, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions.
Sequences containing transcription terminators mark the end of the gene for transcription machinery. This element is most important when very long non-coding sequences follow the end of the mRNA coding region or circular plasmid templates. In addition to producing mRNAs with more uniform length and stability, the terminator sequence helps preserve the transcription reaction materials (NTPs) for the synthesis of coding sequences.
the length would be- 40 bp (promoter), initiation site- 100bp, 25bp, termination site 50 bps.
it would have a DNA polymerase, promoters, stop codons dntps etc.
Amatoxin binds to RNA polymerase II, one of the enzymes responsible for expression of genes in human cells. these are deadly even at low concentrations.
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