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.

Determine the DNA sequence of the 35-bp region examined.

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 digested DNA fragments, with gaps indicating protected regions where proteins are bound.
2. Step 2: Analyze the electrophoresis gel lanes. Lane 1 represents unprotected DNA, showing a complete digestion pattern. Lane 2 represents DNA protected by the repressor protein, and lane 3 represents DNA protected by RNA polymerase. Compare the patterns in these lanes to identify protected regions.
3. Step 3: Correlate the gel bands with the DNA sequence map. The numbers along the gel correspond to the 35-bp sequence labeled on the map in part (c). Use the absence of bands in lanes 2 and 3 to determine which regions of the DNA sequence are protected by the repressor protein and RNA polymerase.
4. Step 4: Deduce the DNA sequence from the gel. The unprotected DNA in lane 1 provides the full digestion pattern, which can be used to infer the sequence. Combine this information with the protected regions identified in lanes 2 and 3 to reconstruct the 35-bp DNA sequence.
5. Step 5: Verify the sequence alignment. Ensure that the reconstructed DNA sequence matches the map provided in part (c) and aligns with the protection patterns observed in the gel. This confirms the accuracy of the sequence determination.

Key Concepts

DNase I Footprint Analysis

DNase I footprint analysis is a technique used to study protein-DNA interactions. In this method, DNase I is used to digest DNA, but the presence of bound proteins protects certain regions from cleavage. The resulting fragments are then analyzed through electrophoresis, allowing researchers to identify which parts of the DNA were protected by proteins, indicating their binding sites.

Electrophoresis

Electrophoresis is a laboratory technique used to separate macromolecules, such as DNA, based on their size and charge. In this process, DNA 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, allowing for the visualization of different DNA fragments after staining, which is crucial for analyzing the results of DNase I footprinting.

Operon and Transcription Regulation

An operon is a cluster of genes under the control of a single promoter, allowing for coordinated regulation of gene expression. In prokaryotes, transcription factors, such as repressors and activators, bind to specific regions of the operon to regulate transcription. Understanding how these proteins interact with the DNA control region is essential for interpreting the results of the DNase I footprint analysis, as it reveals which sequences are critical for transcription regulation.