The electrophoresis gel shown in part (a) is from a DNase I footprint analysis of an operon transcription control region. DNA sequence analysis of a 35-bp region is shown in part (b). The control region, labeled with ³²P at one end, is shown in a map in part (c). Separate samples of control-region DNA are exposed to DNase I, and the resulting DNase I–digested DNA is run in separate lanes of the electrophoresis gel. Unprotected DNA is in lane 1, DNA protected by repressor protein is in lane 2, and RNA polymerase–protected DNA is in lane 3. The numbers along the electrophoresis gel correspond to the 35-bp sequence labeled on the map in part (c). Use the information provided to solve the following problems.

Locate the regions of the sequence protected by repressor protein and by RNA polymerase.

Verified step by step guidance

1. Step 1: Understand the DNase I footprinting technique. DNase I footprinting is used to identify regions of DNA that are bound by proteins, as these regions are protected from DNase I digestion. The electrophoresis gel shows the DNA fragments generated after DNase I digestion, with gaps in the gel indicating protected regions where proteins are bound.
2. Step 2: Analyze lane 1 (unprotected DNA). This lane serves as a control and shows the pattern of DNA fragments when no protein is bound to the DNA. It provides a baseline for comparison with lanes 2 and 3.
3. Step 3: Examine lane 2 (DNA protected by repressor protein). Identify gaps or missing bands in the gel compared to lane 1. These gaps correspond to regions of the DNA sequence that are protected by the repressor protein, as DNase I cannot digest DNA bound by the protein.
4. Step 4: Examine lane 3 (DNA protected by RNA polymerase). Similarly, identify gaps or missing bands in the gel compared to lane 1. These gaps correspond to regions of the DNA sequence that are protected by RNA polymerase, indicating where the polymerase is bound to the DNA.
5. Step 5: Map the protected regions to the 35-bp sequence. Use the numbers along the gel and the sequence map provided in part (c) to locate the specific nucleotide positions that are protected by the repressor protein and RNA polymerase. This will allow you to determine the exact regions of the sequence involved in transcription regulation.

Key Concepts

DNase I Footprint Analysis

DNase I footprint analysis is a technique used to identify the specific regions of DNA that are bound by proteins, such as transcription factors. When DNA is treated with DNase I, the enzyme cleaves unprotected regions, while bound proteins shield their respective DNA segments from digestion. The resulting fragments are then analyzed through electrophoresis, allowing researchers to visualize which parts of the DNA are protected by proteins, indicating their binding sites.

Electrophoresis

Electrophoresis is a laboratory method used to separate DNA, RNA, or proteins based on their size and charge. In this process, samples are placed in a gel matrix and subjected to an electric field, causing the molecules to migrate. Smaller fragments move faster and farther than larger ones, creating distinct bands that can be visualized. This technique is crucial for analyzing the results of DNase I footprinting, as it allows for the comparison of protected versus unprotected DNA segments.

Transcription Regulation

Transcription regulation refers to the mechanisms that control the transcription of genes, determining when and how much of a gene product is produced. Proteins such as repressors and activators bind to specific regions of DNA, influencing the recruitment of RNA polymerase, the enzyme responsible for synthesizing RNA from a DNA template. Understanding how these proteins interact with DNA is essential for interpreting the results of footprinting experiments, as it reveals the functional elements of gene control regions.