Existence Predicted of Messenger RNA: The Operon Model Until 1960 it was thought that rRNA represented the set of templates for protein synthesis. Jacob and Monod questioned this idea because rRNAs have homogenous sizes (5S, 16S, and 23S in bacteria) whereas proteins are of heterogeneous size. From the analysis of mutant bacteria which were altered in their ability to control lactose metabolism, they correctly predicted the existence of mRNA. Bacterial Lactose Metabolism: Lactose use is controlled in bacteria by three enzymes whose genes are adjacent on the chromosome (operon), one of these proteins is galactosidase which hydrolyzes lactose and other -galactosides. - When grown on glucose as a energy source- lactose enzymes are very low in bacteria. - When shifted to lactose rich media- these enzymes are highly expressed. Removal of lactose rapidly leads to very low expression of the proteins. These rapid changes suggested that the template for producing these enzymes was rapidly synthesized on demand, and as rapidly degraded when a continued stimulus (lactose) was absent. Because rRNAs are stable, they reasoned that they were not the intermediates in this information transfer. They used a variety of bacterial mutants: Some expressed the enzymes at high levels in the absence of lactose, others never made these enzymes in the presence of lactose. These results led to the idea of a molecular REPRESSOR that regulated gene expression by binding to a specific OPERATOR sequence on DNA to block RNA synthesis. Based on these studies, they formulated a unifying hypothesis: They proposed that transcription involved copying a DNA strand to give an RNA of complementary sequence, and that this process was controlled at the initiation stage. Hypothetical regulatory elements called repressors and operators controlled the synthesis of other hypothetical RNAs termed messenger RNA.
Regulation of Prokaryotic Transcription: If all cells have the same DNA content, and the DNA of a cell specifies its activities (what enzymes it makes) and characteristics (what effect these enzymes have), why aren't all cells the same? We know that there are different cell types in our bodies, and that the activities of these cells changes with time. How do these cells know which gene products are needed and when they are needed or not needed? This question as it applied to large, complex organisms like humans was very daunting for scientists in the first half of the 20th century. Francois Jacob and Jacques Monod approached the problem from a more basic and simple perspective. How did single-celled prokaryotes like E. coli know how to respond to their environments? Each environmental cue generates a specific response, with specific proteins and reactions. For example, a bacterium can use several different sources of nitrogen. Some bacteria can incorporate diatomic nitrogen gas from the air, or incorporate ammonia from their surroundings, or break the amine group from the end of an amino acid like glutamine. It is much easier and less energy costly for the cell to use the nitrogen from glutamine than to fix nitrogen gas from the air. These two processes require very different enzymes to allow them to occur. If there is glutamine around, the cell should be able to shut off the enzymes that are involved in the incorporation of nitrogen gas. In fact, it shouldn't have to waste the energy to synthesize these enzymes at all (remember how much energy is needed to carry out translation). How can the cell "turn off" the synthesis of proteins from its DNA, when the moment calls for it? How are RNA levels regulated? Primarily regulated by controlling how frequently each gene is transcribed to RNA. Initiation of Transcription is the major control point:
So the central question is: What determines the frequency at which a specific gene is transcribed? The Transcription rate is controlled by proteins that bind to specific DNA sequences in the promoters of a particular gene. Promoter sequences are usually found at the 5' end of a gene relative to the coding region. These proteins are called "Transcription Factors". Each recognizes a particular DNA sequence.
Binding of a transcription factor to a promoter sequence determines whether or not RNA polymerase binds to and initiates transcription of a particular gene.
Methods for regulating gene expression: The cell could somehow selectively inhibit transcription of the gene. The mRNA for this gene would never be made. The cell could selectively degrade the mRNA as soon as it was made, preventing it from being translated into protein. The cell could selectively prevent translation on an otherwise stable mRNA. The cell could selectively degrade the translated protein so that it couldn't waste energy trying to catalyze reactions that the cell has no need for at that time.
In all these cases, the cell has to have some way of shutting off the unwanted protein selectively and leaving on the other genes in the cell. As you can imagine, in terms of energy cost, it is better to shut off the process as early as possible, so that no energy is wasted in mRNA and protein synthesis. This type of early-intervention control is called transcriptional regulation, since expression of the gene is regulated at the level of mRNA synthesis, or transcription. Jacob and Monod were the first scientists to elucidate a transcriptionally regulated system. They worked on the lactose metabolism system in E. Coli. When the bacterium is in an environment that contains lactose:
It should turn on the enzymes that are required for lactose degradation. These enzymes are: beta-galactosidase: This enzyme hydrolyzes the bond between the two sugars, glucose and galactose. It is coded for by the gene LacZ. Lactose Permease: This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose. It is coded for by the gene LacY.
Thiogalactoside transacetylase: This enzyme catalyzes the acetylation of beta galactosides that cannot be hydrolyzed by beta galactosidase. Once modified these molecules can diffuse through the plasma membrane and are eliminated from the cell. LacA These three enzymes appear adjacent to each other on the E. Coli genome. They are preceded by a region that is responsible for the regulation of the lactose metabolic genes. These genes are thus part of an Operon that is transcribed from a single promoter to produce a large mRNA molecule containing three separate protein-coding regions. It would seem that the cell would want to turn these genes on when there is lactose around and off when lactose is absent. But we will see that regulation is more complicated than that (even for a very simple system in a "simple" organism). A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. So if both glucose and lactose are available, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism. There are sites upstream of the Lac genes that respond to glucose concentration.
This assortment of genes and their regulatory regions is called the Lac operon.
Element Operator (LacO) Promoter (LacP) Repressor (LacI) binding Pi CAP
Purpose Binding site for repressor Binding site for RNA polymerase Gene encoding lac repressor protein. Binds to DNA at operator and blocks of RNA polymerase at promoter Promoter for LacI Binding site for cAMP/CAP complex
The LAC Operon: When lactose is present, it acts as an inducer of the operon. It enters the cell and is converted to allolactose in a reaction catalyzed by -galactosidase. The allolactose form of lactose is the actual inducer and binds to the Lac repressor, causing a conformational change that allows the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA and RNA can be made from the three genes. Lactose can now be metabolized.
When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made.
Note: RNA polymerase can still bind to the promoter although it is unable to past move it because of the binding of LacI. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription; the promoter doesn't have to wait for the holoenzyme to bind. We could say that the operon is primed for transcription upon the addition of lactose. It is also important to remember that LacI does not bind to DNA (Lac operator) with infinite affinity. Even in the absence of allolactose, LacI will release the operator. Although another molecule of LacI will quickly bound the operator site, a very brief period of transcription will result.
This allows the cell to continue to produce the small amount of galactosidase which is needed if the cell is to respond to lactose in the environment (to make allolactose).
When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. So when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, by increasing the affinity of the site for RNA polymerase (makes the promoter stronger). This phenomenon is called catabolite repression, a misnomer since it involves activation, but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons.
Methods for Studying Regulation Now 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 allolactose 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 -galactosidase activity. This is very easy, because -galactosidase can be fooled into cleaving a colorless substrate called ONPG into a yellow product called ONP. 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 allolactose is present. Genotype wild type LacZLacPLacOLacILacI(s) LacO-LacI(s) Cis and Trans TEST
There are two different kinds of elements present in the lac operon's regulation. There are DNA binding sites (LacP, LacO, Pi), and proteins (CAP, LacI, 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 a DNA-binding site mutant from a mutant protein? We can use 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.
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Phenotype +IPTG -IPTG + + + + + +
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 (we can then measure the production of element 2 in our assays). 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).
Strategies for Understanding Regulation 1. Find mutations t...