Ch. 8 - Molecular Biology of Transcription and RNA Processing

Sanders - Genetic Analysis: An Integrated Approach 3rd Edition

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Sanders 3rd Edition

Ch. 8 - Molecular Biology of Transcription and RNA Processing

Problem 19b

Chapter 8, Problem 19b

A 1.0-kb DNA fragment from the end of the mouse gene described in the previous problem is examined by DNA footprint protection analysis. Two samples are end-labeled with ³²P, and one of the two is mixed with TFIIB, TFIID, and RNA polymerase II. The DNA exposed to these proteins is run in the right-hand lane of the gel shown below and the control DNA is run in the left-hand. Both DNA samples are treated with DNase I before running the samples on the electrophoresis gel.
Draw a diagram of this DNA fragment bound by the transcriptional proteins, showing the approximate position of proteins along the fragment.

Verified step by step guidance

Step 1: Understand the DNA footprint protection analysis technique. This method identifies regions of DNA that are bound by proteins, protecting them from DNase I digestion. The protected regions appear as gaps or 'footprints' in the gel electrophoresis pattern.

Step 2: Analyze the gel results. Compare the left-hand lane (control DNA treated with DNase I) and the right-hand lane (DNA treated with DNase I after being bound by TFIIB, TFIID, and RNA polymerase II). Identify regions in the right-hand lane where bands are missing or reduced, indicating protein binding.

Step 3: Determine the approximate positions of the proteins along the DNA fragment. Use the gel pattern to map the protected regions to specific locations on the 1.0-kb DNA fragment. These regions correspond to the binding sites of TFIIB, TFIID, and RNA polymerase II.

Step 4: Draw the diagram. Represent the DNA fragment as a linear sequence and mark the positions of the proteins (TFIIB, TFIID, RNA polymerase II) based on the gel analysis. Use symbols or labels to indicate the proteins and their binding sites.

Step 5: Ensure the diagram follows the illustration style seen in Research Technique 8.1. Include clear labels, a scale for the DNA fragment (e.g., kb markers), and visually distinguish the protein-bound regions from unbound regions.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

DNA Footprint Protection Analysis

DNA footprint protection analysis is a technique used to identify the specific binding sites of proteins on DNA. When proteins bind to DNA, they protect certain regions from cleavage by DNase I, allowing researchers to determine where these proteins interact with the DNA. The resulting pattern, observed through gel electrophoresis, reveals the protected areas, indicating the locations of protein binding.

Transcription Factors

Transcription factors are proteins that bind to specific DNA sequences to regulate gene expression. In this context, TFIIB and TFIID are essential transcription factors that assist in the formation of the transcription initiation complex with RNA polymerase II. Understanding their roles helps in visualizing how they interact with the DNA fragment during transcription initiation.

Electrophoresis

Electrophoresis is a laboratory technique used to separate DNA fragments based on their size and charge. In this experiment, the DNA samples are subjected to an electric field, causing the fragments to migrate through a gel matrix. The distance traveled by each fragment allows researchers to analyze the presence of bound proteins and the size of the protected DNA regions, providing insights into protein-DNA interactions.

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Textbook Question

Wild-type E. coli grows best at 37°C but can grow efficiently up to 42°C. An E. coli strain has a mutation of the sigma subunit that results in an RNA polymerase holoenzyme that is stable and transcribes at wild-type levels at 37°C. The mutant holoenzyme is progressively destabilized as the temperature is raised, and it completely denatures and ceases to carry out transcription at 42°C. Relative to wild-type growth, characterize the ability of the mutant strain to carry out transcription at 40°C

Textbook Question

Wild-type E. coli grow best at 37°C but can grow efficiently up to 42°C. An E. coli strain has a mutation of the sigma subunit that results in an RNA polymerase holoenzyme that is stable and transcribes at wild-type levels at 37°C. The mutant holoenzyme is progressively destabilized as the temperature is raised, and it completely denatures and ceases to carry out transcription at 42°C. Relative to wild-type growth, characterize the ability of the mutant strain to carry out transcription at 37°C

Textbook Question

A 3.5-kb segment of DNA containing the complete sequence of a mouse gene is available. The DNA segment contains the promoter sequence and extends beyond the polyadenylation site of the gene. The DNA is studied by band shift assay, and the following gel bands are observed.

Match these conditions to a specific lane of the gel.

3.5-kb fragment plus TFIIB

Textbook Question

A 1.0-kb DNA fragment from the end of the mouse gene described in the previous problem is examined by DNA footprint protection analysis. Two samples are end-labeled with ³²P and one of the two is mixed with TFIIB, TFIID, and RNA polymerase II. The DNA exposed to these proteins is run in the right-hand lane of the gel shown below and the control DNA is run in the left-hand. Both DNA samples are treated with DNase I before running the samples on the electrophoresis gel.

What length of DNA is bound by the transcriptional proteins? Explain how the gel results support this interpretation.

Textbook Question

Explain the role of DNase I.

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Textbook Question

Match these conditions to a specific lane of the gel.

3.5-kb fragment plus RNA polymerase II

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