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The lac operon. In bacteria, the genes involved in the same process are often clustered together. For example, the genes that allow E. coli to break down.

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Presentation on theme: "The lac operon. In bacteria, the genes involved in the same process are often clustered together. For example, the genes that allow E. coli to break down."— Presentation transcript:

1 The lac operon

2 In bacteria, the genes involved in the same process are often clustered together. For example, the genes that allow E. coli to break down milk sugar (lactose) to produce energy. Lactose catabolism

3 lacY encodes lactose permease - transports lactose into the cell lacZ encodes -galactosidase – enzyme that catalyses the reaction: lactose glucose + galactose lacA encodes lactose transacetylase – biological function unclear. Lactose catabolism

4 These genes are controlled. E. coli is a successful competitor in the gut because it doesnt waste time and energy making mRNA and proteins that are not needed. The lac genes are only transcribed if lactose is present in the growth medium. These genes are expressed co-ordinately. Either they are all switched on or they are all switched off. Lactose catabolism

5 The coordinate regulation arises from the clustering of the genes (strictly called CISTRONS) into a structure called an OPERON. There is also a regulatory gene, the lacI gene, that is not part of the operon. This produces a repressor protein that controls the operon. Lactose catabolism

6 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA

7 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA The three coding sequences lie side by side but there is only one promoter

8 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA This means that there is only one mRNA that encodes three proteins. Each coding region has its own start and stop codon

9 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA The separate lacI gene is not controlled. It has Its own promoter and encodes a repressor protein. It is not part of the operon

10 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA In the absence of lactose, the repressor protein binds to a special site in the operon called the OPERATOR and prevents RNA polymerase from moving along the DNA

11 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA In the presence of lactose (effector), the repressor protein binds to the lactose and changes shape. It now falls off the operator and RNA polymerase can transcribe the operon

12 DNA LacI repressor RNA polymerase Active repressor binds to operator Lactose absent: operon switched off The lac operon mRNA P lacI P lacO lacZ lacY lacA

13 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) lactose LacI repressor Inactive repressor- effector complex RNA polymerase P lacI P lacO lacZ lacY lacA The lac operon

14 François Jacob and Jacques Monod worked out how the lac operon functioned and they formulated the operon hypothesis. Jacob Monod Jacob and Monod The lac operon

15 The properties of various mutants allowed Jacob and Monod to work out how operons work. lac mutants The lac operon

16 P O lacZ lacY lacA Active -galactosidase enzyme DNA mRNA protein DNA X X X mRNA protein lacZ mutations are recessive The lac operon Lactose catabolism

17 Mutants where the lacI gene has mutated, will grow on lactose. However they make β-galactosidase all of the time. These mutants that have lost the ability to control gene expression are called constitutive mutants. They are also recessive. Constitutive mutants The lac operon

18 DNA LacI repressor RNA polymerase Active repressor binds to both operators X X No active repressor to bind to operator P lacI P lacO lacZ lacY lacA The lac operon lacI mutations are recessive

19 Jacob & Monod realised that if their operon hypothesis was right, there should be another type of constitutive mutant – one where the operator has mutated so that the repressor cannot recognise it. Such mutants should be dominant and it should be possible to isolate them in a diploid. A testable prediction The lac operon

20 DNA mRNA LacI repressor RNA polymerase Active repressor binds only to wild type operator X DNA lacO c mutations are dominant The lac operon P lacI P lacO lacZ lacY lacA

21 Jacob & Monod mutated a diploid wild type to see whether they could get constitutive mutants. They did get them, and showed that they mapped to the operator region. This supported their hypothesis. The lac operon

22 The lac repressor is an example of a negative regulatory protein, whose action prevents expression of the genes under its control and whose function is controlled by an effector molecule (in this case, lactose).

23 The lac operon Catabolic repression The lac operon is also under the control of a positive regulatory protein. E. colis preferred carbon source is glucose. Glucose inhibits transcription of the lac operon, even in the presence of lactose. Inhibition occurs in lacI and lacO mutants, as well as wild type, indicating the effect of glucose is NOT via the repressor-operator interaction.

24 The lac operon The effect of glucose is mediated by a nucleotide, cyclic AMP (cAMP). The intracellular concentration of cAMP is high in the absence of glucose and low in its presence. cAMP binds to a catabolic activator protein (CAP), upstream of the lac promoter driving the lac operon. When bound to cAMP, CAP enhances lac transcription.

25 Transcription translation -galactosidase enzyme lactose permease lactose trans- acetylase DNA mRNA protein (polycistronic message) Glucose RNA polymerase The lac operon P lacO lacZ lacY lacA cAMP CAP

26 The lac operon Regulation of expression of the lac operon is under two sets of controls, both of which are governed by environmental factors. The repressor-operator interaction provides an all or none level of control (lactose on). [CAP-cAMP]-CAP-binding site interaction provides a modulatory control. (glucose levels control rate of mRNA initiation) Summary

27 Complementation Diploid, Haploid Dominant, Recessive Homozygous, Heterozygous Cistron Cross-feeding

28 Colinearity of the gene and protein Protein structure Haemoglobin Genetic code amino acid sequence

29 The Genetic code Codon Dictionary of the genetic code How the code was deciphered How the code works

30 tRNA and Translation RNA translation (5' 3') Ribosomes Structure of tRNA Anticodon Mechanism of translation Wobble hypothesis

31 Inosine (I) is a rare base found in tRNA, often in the anticodon, capable of binding to adenine, uracil or cytosine.

32 RNA Translation RNA growth (5' 3') RNA polymerase, structure and properties Promoter, consensus Mechanism of translation Termination

33 Suggested reading Regulation of gene transcription (2000) In: An Introduction to Genetic Analysis. pp Griffiths, A. J. F,. Miller, J. H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W. M. (Eds). Freeman and Company, New York.


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