CELL BIOLOGY 220 JMU

CELL AND MOLECULAR BIOLOGY - BIO 214

ASSIGNMENT OF THE WEEK: #7

Mutations affecting eukaryotic gene expression - with a twist.

Introduction

In eukaryotic cells most genes are not transcribed unless signals are present that turn transcription on. Signals vary widely and can be environmental (e.g. light, temperature, etc.), or internal (e.g. hormones). Each of these signals must eventually transmit information to proteins called "transcription factors" that bind to DNA in the promoters of the genes to be turned on. This overall process is called signal transduction. In contrast, genes which are always transcribed and translated (unregulated) are said to be expressed "constitutively".

Within the promoters of genes there are short sequences that serve as binding sites for transcription factors (TF). Some TFs bind to similar sequences in the promoters of many genes. One such common transcription factor is TFIID that binds to the "TATA box" which is present in almost all promoters. The TATA box is located only 20-35 nucleotides upstream from the start site for transcription. In contrast, other transcription factors bind to specific regions present in only one or a few genes. These specific binding sites are called, enhancer elements and are located farther from the transcription start site, sometimes up to thousands of nucleotides in either direction!

What regulates TF binding?

Only when the transcription factors are present and capable of binding to a promoter element is transcription of the downstream gene turned on. There are two basic mechanisms for converting TFs from an inactive state (incapable of binding to DNA) to an active state in which the will bind to a promoter.

1. For some TFs the presence or absence of small molecules within the cell that bind to the TF determines whether or not the TF binds to the promoter element. For example, some steroid hormones when present in a cell at a high enough concentration induce the expression of certain genes by binding to, and changing the conformation of, specific TFs. Without the hormone the transcription factor cannot bind to the promoter. Likewise, without the transcription factor, the hormone would have no effect.

2. Other transcription factors only bind to enhancer elements after they have been modified. One type of modification is phosphorylation in which a phosphate group from ATP is attached to a TF by a specific protein kinase. The phosphate could then be removed by a specific protein phosphatase. Whether the TF is phosphorylated or not would thus depend on the relative activities in the cell of the kinase and the phosphatase. Regulation of the expression and activities of the kinases and phosphatases is obviously important in understanding the entire system...

How is gene regulation studied?

Biologists often use naturally occurring or induced mutations to study these complex systems. Suppose you were interested in the regulation of an certain enzyme. You might be able to detect the presence of that enzyme in your favorite organism, such as the model plant Arabidopsis thaliana, by using a specific enzyme assay. By treating seeds of Arabidopsis with chemicals such as ethyl methane sulfonate that introduce random point mutations into the genome you can generate a large number of mutagenized individuals. By examining many of these individuals using the enzyme assay, you might be lucky enough to find a few that have altered levels of enzyme activity. These mutants will contain nucleotide changes in any of the many components of the signal transduction chain regulating the activity of your enzyme. As you will see, not all of the mutations will be in the gene encoding the enzyme!

The assignment this week is to make a list of all of the possible types of mutations and their effects (positive or negative) on the production and function of a hypothetical gene product called "Learnin". Consider that the Learnin gene is turned on by a transcription factor called "Monroe". Monroe only binds to the Learnin promoter when it is activated by "eye contact" with an enthusiastic student. The gene encoding the Monroe transcription factor is itself turned on by a different transcription factor "salary" but for simplicity we will assume that the salary level is constant. Mutations in the Monroe promoter, however, could affect the ability of salary to bind. Think about all of the possible mutations in either gene (monroe and learnin) that could effect the rate of Learnin activity. Remember that small molecules (i.e. eye contact) and proteins (i.e. monroe) cannot be mutated, only DNA can be mutated. Most of these mutations will affect the binding of any two components in either a positive or negative way. For example, mutations might cause binding to be so tight that it is essentially irreversible or they might prevent binding altogether. Print the following answer form on which a model of the system has been drawn.

Answer one is filled out as a sample. Can you come up with 8 other types of mutations that affect Learnin? Would any mutations result in constitutive expression of Learnin? (distance learnin?)