1. THE LAC OPERON BEGINS

2. Hello there boys and girls. I’m Mr. Milk and I’m here to present the Lac Operon. The Lac Operon is found in bacteria and basically acts like and on –off switch for the bacteria genes. Operons are special because normally, each gene has its own promoter. Here several genes required for function, but only have the one promoter Do you see the agar plates with the bacteria growing on them? Those bacteria will only make the enzyme to break down lactose is lactose is added This bacteria is on a medium with minimal nutrients and displays limited growth. The bacteria are not producing lactose digesting enzymes. If the same petri dish is flooded with lactose, the bacteria will turn on the gene to make the enzyme to digest lactose. They grow and divide to fill the entire dish. Note: lactose cannot be used for cellular metabolism unless it is broken into glucose and galactose – glucose into glycolysis

3. There are names for particular agar broth mixtures that are used to test for bacteria with specific functional genes and thus proteins. It is one of the mechanisms of bacteria identification in lab cultures. MacConkey lactose and tetrazolium lactose media both both contain lactose and bacteria that grow on these plates have to have the enzyme β-galactosidase. That includes lac Z and Y. This is the process of creating streak plates of bacteria. The agar may contain various nutrients that can be used to identify if the bacteria will grow on the media. If colonies of the sample are seen growing, they can use the media. If no sample colonies are seen, the bacteria cannot utilize the nutrient resource.

4. If you put some of those bacteria into my milk container, they will also turn on the gene to produce the lactose digesting enzymes. How do the bacteria know to turn on that gene? They don’t have brains, yet it seems that they are making a decision on which enzymes to manufacture inside their cells. In bacteria, as well as other organisms, this is called REGULATION of gene expression. Genes that produce specific proteins must turn on or off at the right times and in the right places. The Lac operon is a study in bacteria (a simple system) used to improve our understanding of how this occurs. Eukaryotic Gene Regulation is very complicated and will be covered in another presentation. The bacteria utilize something called an operon to control gene expression. They can turn on or off the lactose digesting gene with the LAC OPERON. Let me explain how it works Jacob and Monad (France)conducted this experiment with E coli bacteria. They researched the Lac operon model we will study. They proposed that genes with related functions are grouped together as units called operons. There are others such as the tryptophan operon etc. Hope you still remember milk contains lactose!

5. First we must start with the DNA. DNA is not just pure DNA on the chromosome. Rather it is associated with vast amounts of protein which is involved in control of the genes on the DNA. In our model of the Lac Operon, we will represent some of the associations with DNA and protein by naming the DNA regions. In order for DNA to be transcribed, it must have a region (called the promoter) for the RNA polymerase to join. Promoter RNA polymerase RNAp DNA CODE -3’  5’ RNA p Transcribed to mRNA ex 5’AUGGGCGGAAAUUUGGA3’ Translated to amino acids in sequence to make a protein

6. In the process below the repressor protein is made Promoter RNA polymerase RNAp DNA CODE -3’  5’ RNA p Transcribed to mRNA ex 5’AUGGGCGGAAAUUUGGA3’ Translated to amino acids in sequence to make repressor protein – simulated molecular shape below One of the most important aspects of the operon is the control system. The lac operon utilizes a repressor protein that stops the transcription of the lac operon by blocking the gene access by RNA polymerase. We’ll see how this is done later. Repressor protein Note there is a codon but no tRNA for the stop signal.

7. The repressor protein has a specific shape that bonds with a part of the DNA on the Lac operon called the OPERATOR. The operator is locate d in between the PROMOTER and the STRUCTURAL gene of the lactose breakdown genes. Repressor protein Operator Lactose breakdown enzyme genes Promoter RNA p Hey, I’m blocked. I can’t engage the DNA to begin any copy process! The lactose enzyme information is not transcribed. No lactose breakdown enzymes are made. This gene is effectively turned OFF

8. The lac operon is an INDUCIBLE Operon. Addition of an inducer(signal molecule) turns the gene ON. The inducer is the chemical that needs to be digested. In this case lactose is the inducer. The inducer bonds to the repressor! Repressor protein Operator Lactose breakdown enzyme genes Promoter RNA p Hey, I’m blocked. I can’t engage the DNA to block RNA polymerase! The lactose enzyme information is transcribed. Lactose breakdown enzymes are made. This gene is effectively turned ON Lactose Transcribed to mRNA ex 5’AUG..3’is called polycistronic –many genes 1 mRNA Translated to amino acids in sequence to make LACOSE DIGEST ENZ

9. There are actually three enzyme breakdown genes of the Lac operon. They are shown below in their 3-D model structures. Each will participate in the breakdown of lactose into the two simple sugars glucose and galactose Remember, lactose is only useful to the bacteria if they can break it down and put the sugars into glycolysis. Glucose enters easily. Galactose is just an isomer of glucose and can be converted. Isomers again. See I told you it would be important! Coded for by Lac A part of operon.Bacteria are capable of catabolism of lactose even without this enzyme Coded for by Lac Z part of operon Coded for by Lac Y part of operon

10. Repressor protein Operator Lactose breakdown enzyme genes Promoter RNA p Hey, I’m blocked again. I can’t engage the DNA to begin any copy process! There is no need to make lactose breakdown enzymes anymore. No more lactose is left. This gene is effectively turned OFF Now once the enzymes are made by the process of transcription and translation, the enzymes start doing their job breaking down the lactose. They continue to do this until all the lactose is used up. When this happens the bacteria then shut off the gene again. ENZYMES LACTOSEGLUCOSEGALACTOSE NO MORE LACTOSENO MORE NEED FOR ENZYMES NO MORE INDUCER TO BLOCK REPRESSOR

11. And that’s the story of how an INDUCIBLE OPERON works. Here is an example using different shape representation The repressor gene is technically not part of the operon and may be physically separated by distance from the operon. This region is all part of the Lac operon which codes for three protein enzymes.

12. Above is another diagram of the lac operon. Once the bacteria are exposed to different sugars, their operons have to either produce enzymes or not. Some control of the Lac operon is due to cAMP –cyclic Adenosine Monophosphate. cAMP is made when glucose levels are low and it acts to TURN ON or ACTIVATE the Lac operon. The Lac operon, however, will still not turn on unless lactose is present. In this case two conditions must be met to turn on the operon. Cyclic AMP is found in both prokaryotes and eukaryotes. It is derived from ATP but is not used for energy but rather as a secondary messenger to control cell activities.(signal transduction)

13. CAP is catabolite activator protein and will greatly increase the production of galactosidase in the absence of glucose. It needs cAMP to activate. Lac operon turned ON when glucose low (high cAMP) and lactose present

14. Some operons work differently than the Lac operon. These are called Repressible Operons. The example is the Trp OPERON In the Trp operon, tryptophan is synthesized because the gene is always turned on unless the inducer is present. Normally, the gene is turned off because tryptophan is present and this bonds to the repressor (tryptophan- repressor complex) changing its shape ( an allosteric change) and causing it to bind to the operator(active repressor). It is a repressible operon because if typtophan is not present, the gene will be on. The repressor is not the right shape to bond to the operator unless the co-repressor(trp) is present REPRESSIBLE - NO INDUCER – STRUCTURAL GENE IS TURNED ON!! Opposite to inducible operon –

15. Trp operon includes 5 genes –make 5 enzymes that convert molecules into tryptophan Bacteria need tryptophan to make cell proteins ( E. Coli) and needs a constant supply. Tryptophan making genes are always turned on by the operon, but making typtophan takes energy If the bacteria find tryptophan in the environment, why would they use energy making something that is readily available? They shut the operon off! The trp operon a repressible operon because it is always ON, but it can be turned OFF. This is accomplished by having the repressor only activated into the matching shape of the operator if tryptophan is present. This shuts the genes off!

16. So the tryp operon is different because it is always on unless tryptophan can be found in the environment. If tryptophan is available, it bonds to the repressor making the repressor have the correct shape to bond to the operator and block the transcription of the trp genes. This is shown by the diagram below. Trp is called a corepressor! It is necessary for it to bond to the repressor for the repressor to engage the operator. Trp is not an inducer!

17. Lets try some questions. In what organisms do you find operons? THINK THINK THINK E. Coli is a bacteria Operons are located in bacteria genomes All prokarytotes contain operons Operons are only found in the prokaryotes Regarding the trp operon; w hen tryptophan is not present, normal transcription continues. Is it an example of negative or positive feedback? This is an example of negative feedback THINK THINK THINK Trp operon is a repressible operon When trp is present genes turn off transcription When trp is absent genes turn on transcription Negative feedback means what is produced accumulates and shuts down the system.

18. First Jacob and Monod grew the bacteria on a culture media containing radioactive amino acids. Then Jacob and Monod added lactose to a culture of E.coli cells, and found that lactose was metabolized. Radioactive amino acids were incorporated into the enzyme beta-galactosidase. This was because the inducer(the lactose) removes the repressor and allows the transcription and translation of the beta-galactosidase gene. The product, β-galactosidase then starts to digest lactose into galactose and glucose. One last thing. Lets take a fast look at the experiment that starting this whole idea of the operon. Remember the French Scientists Jacob and Monod were the ones responsible. Monad in the 1940’s noticed that if glucose and lactose were both provided, glucose would be metabolized first (growth phase I, and then lactose (growth phase II). This phenomenon is called diauxis

19. Lac Operon END Bye for now.