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Prokaryotic Genetics Mutants

Studying Prokaryotic Genetics

Methods for Studying Regulation

Let's think about what mutations in various elements of the Lac operon could exist, and how we see these mutants as experimental scientists. First, here are some notes about how we study the lac operon and some nomenclature for how we describe the bacterial phenotypes that we see:

IPTG (isopropyl-beta-D-thiogalactoside) is a molecule that looks very much like lactose to the Lac repressor (LacI). Thus, this molecule can be used as a gratuitous inducer, because it will induce the Lac operon by altering the conformation of LacI so that it can no longer block the promoter, but it is not a substrate for the lactose metabolism genes.

We can measure the amount of mRNA made on the lac operon (coding lacZ, lacY, and lacA) by measuring the amount of B-galactosidase activity. This is very easy, because B-galactosidase can be fooled into cleaving a colourless substrate called ONPG into a yellow product called ONP. Thus, we can use a replacement substrate with an obvious visible product to measure amounts of the enzyme. If the medium is very yellow, then there is a lot of activity, a lot of mRNA and therefore a lot of transcription activity on the lac operon. We can quantitatively measure the amount of yellow in a spectrophotometer.

A constitutively expressed gene (denoted c) is never turned off. It is making mRNA and protein all the time. An uninducible gene is never turned on. An uninducible DNA binding site is mutated so that it never binds its protein.

A super-repressor can be denoted s. This repressor always represses, regardless of its regulation. For example, a LacI(s) mutant always represses at the promoter regardless of whether or not lactose is present.

Genotype Phenotype
wild type + -
LacZ- - -
LacP- - -
LacO- + +
LacI- + +
LacI(s) - -
LacO-LacI(s) + +

Cis and Trans

What two different kinds of elements are present in the lac operon's regulation? There are DNA binding sites (LacP, LacO, Pi), and proteins (CAP, LacI, and LacZ, LacY, and LacA). When we study these systems, it is useful to be able to differentiate between the two types of elements. For example, look above at the phenotypes of LacO and LacI mutants. They are the same. How would we be able to distinguish which one was a DNA-binding site and which one was a mutant protein?

This is where the pseudo-diploid genetics that we described in the previous section come in handy. We can use these techniques to see if a DNA sequence can act from afar on another DNA sequence. If it can, then it is a diffusible protein. These sites are called trans-acting sites, since they act from afar. If the site cannot act from afar, then it is a DNA binding site that needs to be near other DNA sites (such as coding sequences) in order to function. These sites are called cis-acting sites, since they need to be next to other DNA to work.

In order to see if a DNA element is acting in cis or in trans to another DNA element, we can do a test in which we insert a piece of DNA carrying element 1 into a cell that already has a copy of mutated element 1 next to element 2. If the inserted element can complement or replace the function of the mutated element, it can be said to be acting in trans, since it must diffuse off a plasmid or from another site in the DNA in order to be functional. This, therefore, must be a diffusible protein. On the other hand, if the two functional pieces of DNA must be adjacent to each other to be functional (acting in cis), then one must be a DNA binding site affecting the other.

Therefore we can see that when we do the cis-trans test, any pair of DNA elements that passes both the cis and trans test must be acting in trans (and is therefore a coding region for a protein), and any pair that passes only the cis test must be acting in cis (and is therefore a DNA binding site).

You will learn later that there are some DNA binding elements that can act in trans, through folding and looping of the DNA strand, but these are largely in eukaryotes.

Strategies for Understanding Regulation

  1. Find mutations that render the regulation uninducible or constitutive
  2. Decide by performing a complementation test if the mutants are dominant or recessive
  3. If they are recessive, decide if the system is regulated by repression or by activation. A recessive mutated activator has most likely lost function: the system will become uninducible. A recessive mutated repressor has also lost function, but now the system will have constitutive expression.
  4. Decide if the elements of the system act in cis or in trans to each other: are they diffusible proteins or DNA binding sites?
  5. Construct a model

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