In prokaryote genomes, groups of with related functions are often linked together, with their expression being controlled by a single set of . These gene “bundles” are referred to as operons. Operons are an efficient way to streamline gene expression in prokaryotes. In this module we’ll be looking specifically at the Escherichia coli lac operon, which is often used as a model system in genetics and has real, practical applications in molecular biology. 1. The lac operon1.1 Structure The lac operon contains three enzyme-coding structural genes and three regulatory elements. The enzymes work together to allow E. coli to digest the disaccharide , and the regulatory elements control the transcription of these enzymes. These coding genes always come in a specific order within the operon, and during transcription, they are all transcribed together onto a single strand. Please explore Figure 1 thoroughly by clicking on the “?” icons, to familiarize yourself with the key regulatory elements, structural genes, and protein products of the lac operon.
Figure 1: The lac operonClick on the “?” icons in this Figure to see more information about the component parts of the operon. 2. FunctionWe can see from Figure 1 that the lac operon coordinates the transcription of three enzymes with related functions. This is evidently very practical, but true beauty of this system lies in the fact that it ensures that these genes only get transcribed under specific environmental conditions. Lactose is a relatively rare sugar, and most E. coli don’t need to be producing the beta-galactosidase and permease enzymes at a constant rate. Luckily for E. coli, the lac operon only activates in the presence of lactose! Watch this short video, courtesy of Virtual Cell, to see how this is accomplished: In order to understand this video, you’ll need a good understanding of gene transcription and mRNA translation. If anything in this video seems unfamiliar, please take some time to brush up on these topics. Video Notes:
Hopefully, this video has given you a basic idea of how the lac operon functions. In Section 3, we’ll take a deeper dive into how the individual components of the operon interact with each other by considering what happens if one or more of them is altered by a mutation. 3. MutationsIn molecular biology, one of the most common methods for figuring out a gene’s function is to mutate it and measure the resulting effects on its organism’s phenotype. In this section, we’ll be looking at a variety of mutations that can occur in lac operon genes, and discussing the effects of those mutations on E. coli. To do this, we’ll be using the following symbols to represent the individual components of the lac operon: I P O Z Y A In this model, all the genetic elements in the operon are lined up in the same orientation as they are in an actual E. coli genome (see Figure 1). Since the function of lacA is not yet well defined, we’ll be leaving it out of this model more often than not. When all the sequences are , the lac operon functions normally. We’ll represent this using the following notation: I+ P+ O+ Z+ Y+ A+ If a given gene is mutated, we’ll change the superscript above that gene. Listed below are the specific mutations we are going to be looking at for this course:
In these next exercises, we’ll consider what happens in a typical haploid E. coli when some of these mutations occur. As a hint, remember that all regulatory elements in the operon need to be functioning normally before any structural genes can be transcribed. 4. MerodiploidsTypically, we represent E. coli and other prokaryotes as being completely haploid, with only one circular chromosome and only one copy of each gene. You may remember, however, from our chapter on prokaryote genetics that this isn’t always the case. Bacteria, including E. coli, can acquire DNA from their environment (translation), from phages (transduction) or from other bacteria (conjugation). This may result in E. coli with two copies of certain genes! We call these partially diploid prokaryotes merodiploids (“mero-” comes from the Greek word for “part”, or “partial”). Merodiploids can be produced in a lab setting, using Hfr/F+ strains of E. coli. Merodiploid E. coli are a fantastic research tool. They allow us to examine how wild-type and mutated alleles interact within a living organism, with all the added bonuses of working with E. coli (fast reproduction/growth, easy colony maintenance, etc.) In this module, we’ll be representing merodiploids using the following notation: I+ P+ O+ Z+ Y+ A+ / F’ I+ P+ O+ Z+ Y+ A+ In this notation, we show a chromosomal lac operon and an Hfr plasmid lac operon side by side. Again, we’ve included the lacA gene here for completeness, but will be leaving it out of our exercises. Because merodiploids have two copies of a given set of genes, mutations affect them differently. For example, if a single copy of a protein coding gene is inactivated, the second copy may still continue to produce viable protein, effectively masking the mutation. Try out your understanding using this next set of exercises: 5. Regulators and EffectorsWe’ve seen in Section 2 that the lac operon has a built-in lactose sensor: the repressor protein. When there is no lactose present, the repressor prevents lac operon products from being translated by binding to the operator region. When lactose is plentiful in the environment, it is taken up by the cell and binds to the repressor, removing its ability to bind to the operator region. In general, we call any molecule that modifies a protein’s function in this way an effector molecule. To be a true effector, a molecule must modify a protein’s activity by selectively binding at an . In molecular biology terms, we would say that the repressor protein is a negative regulator of the lac operon, because it’s binding to the operon decreases transcription. In contrast, a positive regulator would be a molecule that binds to the operon and increases transcription. The lac operon does indeed have a positive regulator: Catabolite Activator Protein, or CAP. Keeping pace with the repressor protein, CAP has its own effector molecule: cyclic AMP, or cAMP. cAMP is produced by E. coli as a metabolic byproduct when glucose is scarce. It binds to the allosteric site on CAP, activating the protein and forming what we’ll call the cAMP-CAP complex. Thus activated, CAP binds to the lac operon promoter region, just upstream of the binding site for RNApol. This increases the affinity of the promoter region for RNApol, which leads to a huge increase in lac operon transcription (Figure 2). Without the cAMP-CAP complex, the lac operon is still transcribed in the presence of lactose, but at a much slower rate. Figure 2: The cAMP-CAP complexNow we might wonder, if the lac operon already has a negative regulator, why does it also need a positive regulator? Ultimately, it all comes down to efficiency. E. coli are more efficient at digesting glucose than lactose, so when glucose is plentiful, it’s wasteful to transcribe lac operon enzymes. The most efficient regulatory system would be one which activates not only in the presence of lactose, but also in the absence of glucose; this is what the cAMP-CAP complex accomplishes. Test your understanding using the next set of exercises: A structural gene codes for a product that does not regulate gene expression. Examples include enzymes, structural proteins, siRNA, etc. Regulatory elements are non-coding regions of DNA that function to regulate gene expression. They may contain binding sites for polymerase enzymes, transcription factors, repressor proteins, etc. A disaccharide made up of the two monosaccharides glucose and galactose. A single mRNA strand that contains coding sequences for multiple products. Separate ribosome binding-sites exist for each coding sequence, allowing for simultaneous translation of all sequences. DNA sequences that modify or regulate the expression of distant genes. DNA sequence that modifies or controls the expression of an adjacent gene. An enzyme that transcribes mRNA using DNA as a template The most common form of a gene or phenotype found in nature A binding site other than the protein's active site. In an enzyme, the active site is the site of catalysis. In a DNA-binding protein, the active site is the binding site for DNA. |