Prokaryotes are able to regulate gene expression by applying unique tricks in their toolbox. In prokaryotic cells, genes expression is regulated almost entirely at the level of the initiation of transcription. In eukaryotic cells, regulation of gene expression can occur at all stages of transcription and translation, and even after a protein is made. I will discuss gene regulation in eukaryotes in a future article. For now, I will focus on how prokaryotes regulate gene expression using a mechanism involving the use of a set of genes that share a single promotor, called operons. Before we jump in however, let us look at some key definitions.

Definitions

  1. Gene expression: The process of turning on a gene to produce RNA and protein. A gene is expressed when a protein is made
  2. Operon: A set of genes that share a single promotor and transcribes a single mRNA to make multiple proteins (a polycistronic message)
  3. Operator: A regulatory sequence within DNA that controls the expression of an operon
  4. Promotor: A nucleotide sequence that allows an RNA polymerase to recognize the start of a transcription unit and to initiate transcription
  5. Inducible: An operon can be turned on when an inducer is present
  6. Constitutive: An operon expressed at all times whether inducers are present or not
  7. Allosteric: A change in a protein’s shape that also changes its function

Now with those definitions out of the way, let’s continue building a foundation by appreciating some important naming conventions.

Naming Conventions

  1. Names of DNA sequences are shown in italics e.g. lacZ
  2. The protein product of a gene is put in plain text and sometimes with the letter “p” preceding the gene’s name i.e. placZ
  3. If the version of the gene is functional, it is described with the superscript “+”, i.e., lacZ+
  4. A non-functioning version of the gene is designated with the superscript “-” i.e., lacZ.

Negative Regulation of Gene Expression

The Lac Operon

The Lac operon in prokaryotes is a set of genes controlled by a single promotor that are expressed in response to the presence of lactose in the bacterial cell. The names of the genes in the Lac operon are the Z, Y, and A. They produce three enzymes:

  1. Beta-galactosidase (Z gene): Enzyme that splits lactose into glucose and galactose
  2. Lactose permease (Y gene): A membrane-bound protein that enables uptake of lactose
  3. Transacetylase (A gene): Enzyme that chemically modifies lactose and prevents it from escaping the cell once it enters

All of these proteins work together to metabolize lactose and must all be expressed together. This makes sense. For example, if lactose is present in the environment, lactose permease must be present to get it into the cell, transacetylase must be present to prevent it from leaving the cell, and beta-galactosidase must also be present to digest it down to its constituent monosaccharides.

The Lac operon

The promotor in the Lac operon sits between the -35 to +1 region of the DNA, relative to the transcriptional start site. The operator of the Lac operon is where a regulatory protein called placI can bind and influence the expression of the genes in the Lac operon. The placI protein is coded for by the LacI gene (not part of the LacI operon).

The LacI gene (also known as the lac repressor) is a negative regulator since its protein product (placI) attaches to the operon and negatively affects gene expression. When no lactose (the inducer) is present, placI attaches to the operator, turning it off. This prevent squandering of energy to make enzymes that will not have any work to do.

If lactose is present, lactose binds to placI and causes it to undergo an allosteric change, releasing it from the promotor. Failure to create a “road block” at the promotor allows RNA polymerase to transcribe the downstream genes.

Experiments with Lac Operon Partial Diploids

Attempt the problem below to see if you can think through the outcome of each scenario. If you get these right, you have a very good concept of how the Lac operon works. As you read through the problem remember that a haploid organism has just one copy of its genetic instructions e.g. bacteria. Humans as you know are diploid since we have two copies of our genetic instruction. A partial diploid is a cell that contains two copies of some, but not all of its genes. They are made by inserting plasmids containing the gene of the Lac operon into E. coli.

Problem

Consider the following partial diploid strains of bacteria in the table below. Each of them was grown in media that differ only in their lactose content. Predict whether or not beta-galactosidase will be produced in media 1 (containing no lactose) and in media 2 (containing lactose). Use a plus sign to indicate the ability of the bacteria to metabolize lactose and a minus sign to indicate its inability to metabolize lactose. Note that Oc means that the operator is constitutive i.e., because of a mutation, pLacI is no longer able to bind to the operon’s promotor even when lactose is not present.

Partial Diploid (chromosome/plasmid)Media 1
(lactose absent)
Media 2
(lactose present)
I+Z+/I+Z+
IZ+/I+OcZ+
IOcZ/I+Z+
I+OcZ/I+Z
I+Z/IZ+
Click to find solution

The Trp Operon

Trp in the Trp operon stands for tryptophan. Tryptophan is one of the 20 amino acids used to make proteins. Most bacteria such as E. coli can make tryptophan from scratch. Five proteins must be present for E. coli to make tryptophan. They are coded for by the trpE, trpD, trpC, trpB and trpA genes which all share a single promotor. Just downstream the promotor is the operator on which the ptrpR protein (a repressor protein) can bind.

When tryptophan level is high in the cell, tryptophan binds to ptrpR. This creates an allosteric change that enables it to bind to the operator, blocking transcription of tryptophan the downstream genes by RNA polymerase. This make sense since it would be a useless waste of energy to make tryptophan if it is already freely available within the cell.

When tryptophan levels is low, tryptophan detaches from ptrpR creating a conformational change that prevents it from further binding to the operator. As a result, RNA polymerase is able to transcribe the genes responsible for making tryptophan.

Two other genes are affected by ptrpR. One is the trpR gene itself. When ptrpR is high, it binds to the operator that controls trpR, blocking transcription that would result in production of more ptrpR. This is called autogenous regulation (gene regulating its own expression).

Another gene that ptrpR regulates is the aroH gene. It does this by binding to aroH‘s operator which is located -49 to -29 upstream of the start site. Notice that this region overlaps the promotor site which sits between the -35 and -10 upstream region. The result of the overlap is a kink of the DNA, which prevents the sigma factor on the RNA polymerase from “seeing” the -10. Thus, the kink prevents transcription from proceeding.

Positive Regulation of Transcription

We saw in negative regulation of gene expression that expression is hindered when a protein engages with the DNA. In positive regulation of expression, the opposite happens. Binding of a protein to the DNA promotes gene expression.

An example of this can be seen in the Lac operon. If glucose is present, E. coli would prefer to use it as its main source of energy even if lactose levels are high. How is this accomplished? Take a look at the table below. Notice that the promotor sequences that are recognized by sigma factors is four nucleotides off, compared to the consensus sequence (the sequence that results in the most binding). The differences in the sequence relative to the consensus makes the promotor very weak. That means, it barely gets the attention of RNA polymerase.

This problem is solved in an interesting way. When glucose is scarce, a molecule called cyclic AMP (cAMP) accumulates in the cell. This molecule attaches to a regulatory protein called catabolic activator protein (CAP) which acts as a transcription activator when it binds to the DNA at the CAP site. Once it binds, RNA polymerase has a higher affinity to the DNA, causing transcription rate to be ramped up to metabolize lactose.

When glucose is abundant, there is less cAMP around. Therefore, there are fewer active CAP available. This causes transcription rate to drop. Because the Lac operon promotor is so weak, very little transcription of the Lac operon enzymes take place, even in the presence of lactose. Instead, the cell utilizes the glucose that is available.

Reference: Krane, D. 2021. Bio 2110 Molecular Biology Video Lecture. Wright State University – Lake Campus.

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Courtney Simons
Courtney Simons
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Courtney Simons is a food science professor. He holds a BS degree in food science and a Ph.D. in cereal science from North Dakota State University.