Question

32P-labeled promoter DNA was mixed, in various combinations, with the general transcription factors TFIID (D), TFIIA (A), TFIIB (B), TFIIF (F), and RNA polymerase II (Pol II). Binding of these factors to the promoter DNA was autoradiograph of the gel is shown below. The anode (+ electrode) is at the bottom. Labels on top of each lane indicate the factors mixed with DNA. For binding reactions 3-7 (lanes 3-7), the concentration of RNA Pol II was continually increased from zero (lane 3) to some higher concentration (lane 7). Other factors present in the binding reaction are indicated within the triangle above the lanes. For binding reactions of lanes 8-12, the concentration of TFIIF was decreased zero concentration (lane 12). In experiments of 1lanes 13-15, all factors were added except TFIID (lane 13), TFIIB (lane 14) and TFILIA (lane 15), respectively. The band at the bottom of the gel corresponds assayed in an electrophoretic mobility shift assay. The to free (unbound) DNA (i) At the margin of the autoradiography below, indicate which of the four bands above the naked DNA band (N) corresponds to which complex of DNA with general transcription factors and polymerase. Infer which GTFS are required for Pol II binding to the promoter DNA, and which factors are not required. Justify your conclusion. (ii) In which order bind the required factors to the promoter DNA? Justify your conclusion. (ii) +Pol 8 DeA-B D A B.Pot 4-3- 2- N- 1 2 3 4 56 7 8 9 10 11 12 13 14 15 16

Answer 1

2. Transcription factors play an important role in Transcription of genes in cells . Other than Enzyme RNA pol II for transcription a cell requires general transcription factors for various purposes like helping RNA pol II to bind to promoter, increasing the stability of initiation complex etc.

General Transcription factors bind to target in a proper sequence .

• First of all TFII D comes and binds to promoter DNA sequence like TATA .
• Then TFII A and TFIIB bind to the complex of TFIID and promoter stabilizing it to involve another protein RNA polymerase II .
• TFII F comes to connect RNA polymerase with TATA binding protein of TFIID.

From the autoradiography of the electrophoretic shift mobility assay it is seen in lanes 7, 8 and 9 no free DNA probe was obtained. In lane 7 RNA pol II was in maximum concentration and in lanes 8 and 9 concentration of TFIIF still sufficient to support transcription so complex formation was there between DNA , RNA pol II and GTFs.

In lane 16 also when all factors were present then only no free DNA probe was obtained at bottom showing that complex successful formed in that case.

When we gradually increased supply of RNA pol from lane 3 to 7 we could see DNA bands becoming light . It indicates formation of complex increased as we increased RNA pol concentration from lane 3 onwards .

In lane 12 because TFII F was absent complex formation negatively affected by lack of binding of RNA pol II to promoter so some of DNA probe remained free .

In lanes 13, 14 and 15 due to absence of TF II D, B, and A respectively complex assembly of GTF +RNA pol + DNA affected negatively so DNA remained free .

From overall analysis it could be said that despite lack of TFII F some complex formation was there in lane 12 . So this may be not that necessary in transcription .

Answer. 1 please find below image of answer for question 1

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