Chapter 8, Problem 17P

Summary Introduction

To analyze:

The complete sequence of a gene is present in a $\text{2-kb}$ fragment of E.coli DNA. Transcription of this gene is terminated by the rho protein. The complete promoter sequence as well as the terminator region of the gene is present in this fragment. Band shift assay examined the cloned fragments. Here each lane of a single electrophoresis gel contains the $\text{2-kb}$ cloned fragment under the following conditions:

Lane $\text{1: 2-kb}$ fragment only.

Lane $\text{2: 2-kb}$ fragment plus the core enzyme

Lane $\text{3: 2-kb}$ fragment plus the RNA polymerase holoenzyme

Lane $\text{4: 2-kb}$ fragment plus rho protein

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

b. Identify the relative positions of bands in lane $\text{1 \& 3}$.

c. The relative positions of bands in lanes $\text{1 \& 4}$ are to be explained.

Introduction:

Band shift assay is a technique to study the interactions between protein and DNA. The assay is based on non-denaturing gel electrophoresis that runs complexes of proteins and DNA through it. The migration of either DNA or RNA is compared to the migration of a complex of particular protein with DNA or RNA. This study helps to reveal the degree and nature of binding between them. It is also termed as electrophoretic mobility shift assay. The Band shift assay is the separation technique for proteins, DNA and RNA, the mixture of interest is loaded onto an agarose gel and observed for speed, size, and shape.