complex In different combinations, the transcription factors were added to radio
ID: 252292 • Letter: C
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complex In different combinations, the transcription factors were added to radiolabeled (hot) DNA containing the promoter sequence and promoter proximal sequences, followed by s on a native gel (no Seven different complexes of DNA and protein were observed (numbered 1-7 on the left side of Figure 1). None of the unlabeled (cold) DNA sequences that include a TATAAA sequence. Complexes 2-7 still form in the presence of cold DNA sequences when the cold DNA TATAAA sequence is mutated to TAGAGAA. Complex 1 still does not form when the cold DNA sequence is TAGAGAA The data for the competition experiments with cold DNA are not shown. are formed when the same are performed in the presence of excess amounts of F1I8 TFEE RNA Pol I- complex 7 1 23 4 5 6 7 8 9 10 11 12 Which one of the following best names the technique used in this experiment? A) Western Blot B) DNA Footprinting C) Electrophoretic Mobility Shift Assay (EMSA) DSanger Sequencing E Northern Blot Complex 2 seen in lane 6 is most ikely the protein bound to the radiolabeled DNA A) TFILA B) RNA polymerase Il C) TFII D) TFIIB E) TFIIE Which one of the following is a correct statement about these experiments with DNA and trans acting proteins? The proteins are synthesized at the site where the proteins bind B) The cold DNA and the hot DNA combine to make binding just right. CSome of the unlabeled DNA contains an AT rich area that binds less tightly (two hydrogen bonds nstead of three hydrogen bonds) D) The proteins bound to the radioactive DNA are degraded by the emitted high energy particles so there is no more protein to bind to the unlabeled DNA. E) Some proteins in the transcriptional apparatus do not recognize and/or bind to the promoterExplanation / Answer
The technique used in the experiment is called Electrophorectic Mobility Shift Assay or EMSA. This is an electrophoretic seperation based technique which helps to determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Western blotting is a technique used for detecting protein. DNA footprinting is a method for determining the sequence specificity of DNA-binding proteins. Sanger sequencing is a method of DNA sequencing based on selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication making different lenghts of fragments followed by determination of sequence. Northern blooting is used to detect mRNA.
Comparing lane 3 and 6, 7, 8 and 9 it can be realized that complex 2 seen in lane 6 is most likely the protein TFIIA bound to the radiolabeled DNA sequence. The single band in lane 3 appears when TFIID is present. Comparing 7 and 8 shows the effect of TFIIA which becomes more clear when we see lane 9.The upper band of lane 6 appears only when TFIIA is present in these lanes (see lanes 3,6,7,8 and 9)
The answer for the third question will be 'E', i.e, some proteins in the transcriptional apparatus do not recognize and/or bind to the promoter.
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