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 (see Research Technique 8.1). 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.

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 catalyzing this process. The core enzyme can initiate transcription but requires additional factors, such as sigma factors, to recognize promoter regions. The RNA polymerase holoenzyme includes the core enzyme and these factors, allowing for efficient transcription initiation.

Rho-Dependent Termination

Rho-dependent termination is a mechanism by which transcription is terminated in prokaryotes. The rho protein binds to the RNA transcript and moves along it, eventually catching up to the RNA polymerase. When it reaches the polymerase, it causes the release of the RNA transcript, effectively terminating transcription. This process is crucial for ensuring that genes are expressed correctly and efficiently.

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate DNA fragments based on their size. In this method, an electric current is applied to a gel matrix, causing negatively charged DNA to migrate towards the positive electrode. Smaller fragments move faster and travel further through the gel than larger ones. The resulting banding pattern allows for the visualization and analysis of DNA fragments, which is essential for understanding the effects of various conditions on the cloned DNA fragment.