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 Explain the relative positions of bands in lanes 1 and 4.

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 for the recognition of promoter sequences and initiation of transcription.

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 causing the RNA polymerase to dissociate from the DNA when it reaches a specific termination site, leading to the release of the newly synthesized RNA.

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 presence of proteins, such as RNA polymerase or rho protein, can alter the mobility of DNA fragments in the gel, resulting in different band positions. The relative positions of bands in the lanes indicate how the DNA interacts with these proteins, revealing insights into transcriptional regulation.