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1、Ch. 15 Gene regulation in bacteria,We now understand how genes make proteins, a major step in understanding life. But, there must be a regulation of the process. Why?For multicellular creatures: Does every cell in your body contain IDENTICAL DNA? Yes! Because you were made by mitosis from one origin
2、al zygotic cell. So why are you not just one big ball of identical cells?If you were to isolate mRNAs from different body tissues, you would find different ones!,So.,This means that different genes are turned on or off in different cells, which explains how they come to be different.But even for uni
3、cellular cells including bacteria, there must be regulation, so the cell can adapt to a changing environment. A perfect example of this involves the best understood example of gene regulation there is: the lactose (lac) operon of E. coli.,The lac operon,In bacteria, genes that function in common pat
4、hways are usually clustered together and co-ordinately regulated in a configuration called an operon.The lac operon consists of three structural genes:lacZ, which codes for beta-galactosidaselacY, which codes for lactose permeaseAnd lacA, which codes for transacetylase All of these genes play a role
5、 in the cells ability to metabolize lactose, and use it as a carbon and energy source.,It is easy to experimentally demonstrate two facts:These gene products are not produced in the absence of lactoseE. coli cells prefer glucose to lactose as a carbon source; even if lactose is present, the gene pro
6、ducts will not be made if glucose is also present.When glucose is absent and lactose is present, the lac operon genes are turned on.After all, this makes sense; why go to all the trouble of making proteins that are not going to be used - only make them when they can be used.So how does E. coli turn
7、the genes on and off at the right time?,The structural organization of the lac operon,The three structural genes share a common promoter; another gene, I, codes for a repressor protein, and has its own promoter. An operator region, located near the promoter for the structural genes, binds the repres
8、sor protein under normal circumstances (no lactose).,Under these circumstances, RNA polymerase is blocked from contacting the promoter, so no transcription occurs and the genes are off.,RNAPol,In the presence of lactose (the INDUCER),Lactose binds to the repressor protein, which is allosteric, chang
9、ing its conformation and eliminating its affinity for the operator site. It then dissociates, allowing RNA polymerase to transcribe the genes. As long as lactose is present, the genes will be on.,RNAPol,Mutations in the lac operon,If structural genes are mutated, those proteins dont get made. (Z-, Y
10、-)If the operator is mutated (OC), the regulation is lost, and the gene is always on (since the repressor cannot bind).If the repressor gene is mutated (I-), we get the same effect, since there is no functional repressor to bind the operator.If the structural gene promoter is mutated (P-), no transc
11、ription can take place, because RNAP cannot recognize the promoter.,The wild type condition is written as: Inducer absent /Inducer present b-gal/perm b-gal/permI+P+O+Z+Y+ -/-+/+I+P+O+Z-Y+ -/-/+I+P+OCZ+Y+/+/+I-P+O+Z+Y+/+/+I+P-O+Z+Y+-/-/-What happens if these mutants occur together?P-OCI-I-P-OCZ+Y+-/-
12、/-I-P+OCZ+Y+/+/+,Studies with merodiploids.,Actually proved that I was a trans-acting element (a diffusable protein) while O and P were cis-acting (DNA) elements. Analysis of merodiploid mutants can be challenging.Also, there are some other I mutations:IS = superrepressor - does not recognize lactos
13、e, so it remains bound to the operator, genes OFFI-D = dominance type - affects the part of the repressor responsible for binding polypeptides together (the repressor is a tetramer); it is dominant because it poisons formation of functional repressor even if a good copy of I is present, genes ON.,Ex
14、amples of merodiploid mutants Inducer absent/Inducer present b-gal/perm b-gal/permI-P+O+Z+Y+ -/-+/+I+P+O+Z-Y- I-P+OCZ+Y-+/-+/+I+P+O+Z-Y+ISP+O+Z+Y-/-/-I+P+O+Z-Y+I-P+OCZ-Y+-/+/+I+P+O+Z+Y-Dominance hierarchy:P-OC=I-DISI+I-supplement!,Positive regulation of the lac operon,What we have seen so far is neg
15、ative regulation of an inducible operon.There is also positive regulation going on; it is called catabolite repression or the glucose effect. When glucose is present, the lac genes are not on. How do we account for this?Near the promoter is a site called the CAP site. The CAP protein will bind to th
16、is site if it is bound to cAMP; when bound, it will stimulate transcription.cAMP levels are high when glucose is low, and vice versa.,So, when there is a lot of glucose (even if lactose is present), CAP fails to bind and transcription is not stimulated.Think of it this way:The repressor protein functions as an on/off switchThe CAP/cAMP complex serves as a volume controlNow it is clear why the genes are turned on in the presence of lactose, but only if glucose is absent.The enzyme adenylate cyclase makes cAMP from AMP; if mutated, no cAMP - no stimulation.,
