A 2-kb fragment of E. coli DNA contains the complete sequence of a gene for which transcription is terminated by the rho protein. The fragment contains the complete promoter sequence as well as the terminator region of the gene. The cloned fragment is examined by band shift assay. Each lane of a single electrophoresis gel contains the 2-kb cloned fragment under the following conditions:
Lane 1: 2-kb fragment alone
Lane 2: 2-kb fragment plus the core enzyme
Lane 3: 2-kb fragment plus the RNA polymerase holoenzyme
Lane 4: 2-kb fragment plus rho protein
Explain the relative positions of bands in lanes 1 and 4.

Verified step by step guidance

1. Step 1: Understand the experimental setup. The band shift assay is used to study protein-DNA interactions. In this experiment, the 2-kb DNA fragment is analyzed under different conditions to observe how proteins interact with the DNA and affect its mobility during electrophoresis.
2. Step 2: Analyze Lane 1. This lane contains the 2-kb DNA fragment alone, without any proteins. Since no protein is bound to the DNA, the fragment will migrate based on its size and charge alone, serving as the reference position for comparison with other lanes.
3. Step 3: Analyze Lane 4. This lane contains the 2-kb DNA fragment plus rho protein. Rho protein binds to the terminator region of the DNA, forming a DNA-protein complex. This complex is larger and has altered charge properties compared to the unbound DNA, causing it to migrate more slowly during electrophoresis.
4. Step 4: Compare the relative positions of bands in Lane 1 and Lane 4. The band in Lane 4 will appear higher (closer to the origin) on the gel compared to Lane 1 because the DNA-rho complex has reduced mobility due to its increased size and altered charge.
5. Step 5: Conclude the significance of the band shift. The difference in band positions between Lane 1 and Lane 4 confirms that rho protein binds specifically to the terminator region of the DNA fragment, demonstrating its role in transcription termination.

Key Concepts

Transcription and RNA Polymerase

Transcription is the process by which RNA is synthesized from a DNA template. RNA polymerase is the enzyme responsible for this process, and it exists in two forms: the core enzyme, which can synthesize RNA but cannot initiate transcription, and the holoenzyme, which includes a sigma factor that allows it to bind to promoters and initiate transcription. Understanding how these enzymes interact with DNA is crucial for analyzing the results of the band shift assay.

Rho-Dependent Termination

Rho-dependent termination is a mechanism by which the rho protein facilitates the termination of transcription in prokaryotes. Rho binds to the RNA transcript and moves along it, eventually causing the RNA polymerase to dissociate from the DNA when it reaches a specific termination site. This concept is essential for understanding why the presence of rho protein in lane 4 affects the position of the bands in the gel.

Electrophoresis and Band Shift Assay

Electrophoresis is a technique used to separate nucleic acids based on their size and charge. In a band shift assay, the binding of proteins (like RNA polymerase or rho) to DNA can alter the mobility of the DNA fragment during electrophoresis, resulting in different band positions. Analyzing the relative positions of bands in lanes 1 and 4 helps to determine the effects of protein binding on the DNA fragment.