a) The formation of a hairpin appears to be important for termination of transcr
ID: 14584 • Letter: A
Question
a) The formation of a hairpin appears to be important for termination of transcription in E. coli (both rho-dependent and rho-independent). Design an experiment to test if the hairpin RNA is really important for termination.b) pretend that you have just discovered the Rho protein, and have reason to believe
that it is involved in regulating transcription termination. You hypothesize that rho
might have helicase activity, which can cause the RNA-DNA duplex to disassociate.
Design an experiment to test your hypothesis. You can (and should) consider using
any and all techniques you have learned about in this course (molecular biology)
Thank you! explain if you can so i can understand. :)
Explanation / Answer
a) The simplest way to verify the hairpin's role is to modify the RNA so that a hairpin does not form. A hairpin forms due to the presence of complimentary bases being close together on the same strand. The RNA strand folds on itself and the complimentary bases bind to each other, giving rise to a stable hairpin structure. I would first synthesize DNA that does not encode nearby complimentary bases at its tail end. For example, instead of the DNA reading CCCCATGCGGGG (the RNA's GGGG and CCCC will fold over and bind each other), I would change it to something like CGTAACCGTAG (folding over and self binding is much less likely to occur). This means that the hairpin would not form. I would then add a few amino acid codons after the expected stop sequence. If a hairpin loop is not needed for transcription termination, then bacteria with this modified DNA will terminate as expected and no change in the RNA will be observed. If a hairpin loop is critical, then termination will not occur at the expected site and the resulting RNA will have the additional codons that would not have been included had the hairpin loop been present. The RNA can be extracted from the bacteria and sequenced, revealing the corresponding DNA sequence and transcription termination site. b) I would establish 2 sample groups: a control group with normal rho, dna, and rna an experimental group with modified rho (to prevent activity), normal dna, and normal rna In a sample, add the complimentary single-stranded DNA and RNA and allow to anneal. Next, add the rho protein and incubate. Then, crosslink any DNA-RNA duplexes so that nothing can dissociate. Finally, run the DNA-RNA on a native gel and look for band size. Duplexed DNA or RNA will be much bigger than single-stranded DNA or RNA. Potential results: Control group and experimental group produce gels with small bands only. This indicates that all DNA and RNA was separated prior to gel running. Helicase activity is possibly present, though this is not definitive since protein modification/substitution had no effect on activity. Control group produces small bands, experimental group produces large bands. This means that rho has helicase activity that can be eliminated upon amino acid modification/substitution. Both groups produce large bands. This indicates that rho is unlikely to have helicase activity since none of the DNA-RNA duplexes were separated. Hope this helps some!
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