Eukaryotic Transcription: Deletion analysis of protein binding sequences in a promoter can be difficult to interpret bec

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Eukaryotic Transcription Deletion Analysis Of Protein Binding Sequences In A Promoter Can Be Difficult To Interpret Bec 1 (561.35 KiB) Viewed 78 times

Eukaryotic Transcription: Deletion analysis of protein binding sequences in a promoter can be difficult to interpret because altered spacing between elements can critically affect their function. The linker scanning method eliminates this potential difficulty by replacing 10-nucleotide segments throughout the promoter with oligonucleotide linkers. This method was used to analyze the promoter for the thimidine kinase (Tk) gene. Plasmids containing the Tk promoter in which 10- nucleotide segments had been replaced with linkers were injected into Xenopus laevis oocytes along with control plasmids to measure injection efficiency. Results are shown in the Figure below. 1. Estimate from these results the locations of regions critical for promoter function, and rank their relative importance. Why? 2. Which of these elements do you suppose responds to TBP? Why? 3. What is going on at the start of transcription? positions of linker relative to transcription start site -120 -100 -80 -60 -40 +1 1 1 1 1 1 1 1 10 -20 +20 1 1 1 1 inker-scanned transcript control transcript Figure Legend: Transcripts from linker-scanned plasmids and control plasmids were analyzed by primer extension, using a lio-labeled primer corresponding to sequences about 80 nucleotides from the 5' end of the transcript. These primers were extended to the 5' ends of the transcripts and the products were displayed on a polyacrylamide gel. Two bands are present for both the control and linker-scanned transcripts because of inefficient extension to the very end of the transcript. The position of each linker in indicated at the center of the segment it replaced. A 10-base pair bar is shown in the top left.