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
Diagram the relative positions expected for the DNA fragments in this gel electrophoresis analysis.

Verified step by step guidance

1. Step 1: Understand the experimental setup. The band shift assay is used to study protein-DNA interactions. When proteins bind to DNA, the mobility of the DNA fragment in the gel changes due to the added mass and altered charge. Each lane represents a different condition where the 2-kb DNA fragment interacts with specific proteins.
2. Step 2: Analyze Lane 1. This lane contains the 2-kb DNA fragment alone, which serves as the control. The DNA will migrate to a specific position in the gel based on its size and charge. This position will act as the baseline for comparison with other lanes.
3. Step 3: Analyze Lane 2. This lane contains the 2-kb DNA fragment plus the core enzyme of RNA polymerase. The core enzyme binds nonspecifically to DNA, so the DNA fragment may show a slight shift in mobility due to the added mass of the enzyme. The shift will depend on the strength and specificity of the interaction.
4. Step 4: Analyze Lane 3. This lane contains the 2-kb DNA fragment plus the RNA polymerase holoenzyme. The holoenzyme includes the sigma factor, which allows specific binding to the promoter region of the DNA. Expect a more pronounced shift in mobility compared to Lane 2, as the holoenzyme binds specifically to the promoter sequence.
5. Step 5: Analyze Lane 4. This lane contains the 2-kb DNA fragment plus the rho protein. Rho protein binds to the terminator region of the gene, causing another shift in mobility. The position of the DNA fragment in this lane will reflect the interaction between rho protein and the terminator sequence.

Key Concepts

Gene Transcription

Gene transcription is the process by which a segment of DNA is copied into RNA by the enzyme RNA polymerase. This process involves several stages, including initiation, elongation, and termination. Understanding transcription is crucial for analyzing how genes are expressed and regulated, particularly in the context of the rho-dependent termination mechanism mentioned in the question.

Electrophoresis

Electrophoresis is a laboratory technique used to separate DNA, RNA, or proteins based on their size and charge. In gel electrophoresis, an electric current is applied to a gel matrix, causing the molecules to migrate through the gel. Smaller fragments move faster and farther than larger ones, allowing for the visualization of different DNA fragments based on their length, which is essential for interpreting the results of the experiment described.

Rho Protein Function

Rho protein is a transcription termination factor in prokaryotes that facilitates the termination of RNA synthesis. It binds to the RNA transcript and moves along it, eventually causing RNA polymerase to dissociate from the DNA when it reaches a specific termination site. Understanding the role of rho protein is vital for predicting the outcomes in the electrophoresis analysis, particularly in how it affects the length and presence of RNA transcripts in the gel.