Lac operon

Lac operon - Dr. S. Sivasankara Narayani

1. Y4E01-BASIC MOLECULAR BIOLOGY Dr. s. sivasankara narayani Assistant professor Department of Microbiology Ayya nadar Janaki ammal college sivakasi 12/01/21 Dr.SS ., MRSB (UK)

2. UNIT - I Nucleic acid: DNA as the genetic material – direct and indirect evidences. RNA as the genetic material. Structure, types and properties of DNA and RNA. Chargaff’s rule. Genetic code: Properties – codon - anticodon - *Wobble hypothesis. Law of DNA constancy and C-value paradox. 12/01/21 Dr.SS ., MRSB (UK)

3. UNIT - II DNA replication: Theories - rolling circle mechanism – semiconservative - conservative and discontinuous- evidences. Enzymes involved in replication of DNA in prokaryotes and eukaryotes. 12/01/21 Dr.SS ., MRSB (UK)

4. UNIT - III Transcription process in prokaryotes and eukaryotes: Initiation - elongation - termination. RNA processing in eukaryotes: Capping - polyadenylation - *splicing. Translation in prokaryotes and eukaryotes: Initiation - elongation - termination. Post translational modification in eukaryotes. 12/01/21 Dr.SS ., MRSB (UK)

5. UNIT - IV Regulation of gene expression in bacterial system : Operon concept – lac , trp , ara operon in E. coli - *DNA methylation and heterochromatization . Transposons (brief account only). 12/01/21 Dr.SS ., MRSB (UK)

6. UNIT- V Mutation : Types - molecular basis of spontaneous and induced mutations. Mutagenic agents: Chemical and physical mutagens. Isolation of auxotrophs and drug resistance mutant. Toxicity testing- * Ames test. DNA damage and repair (photoreactivation, excision). 12/01/21 Dr.SS ., MRSB (UK)

7. LAC OPERON The  lac  operon of  E. coli  contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent. Two regulators turn the operon "on" and "off" in response to lactose and glucose levels: the  lac  repressor and catabolite activator protein (CAP). The  lac  repressor  acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting as a repressor when lactose is present. The  lac  repressor senses lactose indirectly, through its isomer  allolactose . Catabolite activator protein  ( CAP ) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule  cAMP . 12/01/21 Dr.SS ., MRSB (UK)

8. OPERON Operon is operating units which can be defined as the cluster of genes located together on the chromosomes & transcribed together. • It is group of closely linked structure genes & associated control gene which regulate the metabolic activity. • All the genes of an operon are coordinately controlled by a mechanism 1st described in 1961 by Francois Jacob & Jaques Monod of the Pasture institute of Paris. 12/01/21 Dr.SS ., MRSB (UK)

9. LAC OPERON The lactose operon designated as lac operon. The lac operon codes for enzymes involved in the catabolism (degradation) of lactose. lactose is the disaccharide which is made up of glucose & galactose. It is the inducible operon since the presence of lactose induce the operon to switched on. 12/01/21 Dr.SS ., MRSB (UK)

10. STRUCTURE The  lac  operon contains three genes:  lacZ ,  lacY , and  lacA . These genes are transcribed as a single mRNA, under control of one promoter. Genes in the  lac  operon specify proteins that help the cell utilize lactose.  lacZ  encodes an enzyme that splits lactose into monosaccharides (single-unit sugars) that can be fed into glycolysis. Similarly,  lacY  encodes a membrane-embedded transporter that helps bring lactose into the cell. In addition to the three genes, the  lac  operon also contains a number of regulatory DNA sequences. These are regions of DNA to which particular regulatory proteins can bind, controlling transcription of the operon. 12/01/21 Dr.SS ., MRSB (UK)

11. . Structural genes of lac operon:   lac Z  codes for the  enzyme β- galactosidase , a tetramer of about 500 kD . This enzyme breaks down β- galactoside into its monosaccharide components. For example,  lactose  is split into  glucose  and  galactose which can be metabolized further through glycolysis. ii.  lac Y  codes for the β- galactoside permease, a 30 kD membrane-bound protein which transports β- galactosides into the cell. iii.  lac A  codes for  β- galactoside transacetylase , that transfers an acetyl group from acetyl-CoA to β- galactosides (role in lac operon not clear). 12/01/21 Dr.SS ., MRSB (UK)

12. Regulatory genes The regulatory genes of lac operon includes  promoter gene, operator gene, lac I , and  catabolite activator protein  (CAP) binding site. The  promoter  is the binding site for  RNA polymerase , the enzyme that performs transcription. The  operator  is a  negative regulatory site  where the lac repressor protein binds. It is located between the promoter and the structural genes and  overlaps  with the  promoter. Lac I (repressor) gene  codes for the lac operon  repressor , which is a tetramer of identical subunits of  38 kD each . This gene is located adjacent to the promoter of the lac operon, with its  own promoter  and  terminator  and is  always  transcribed, hence the repressor is always synthesized. The repressor is a  diffusible  product, making Lac I is a  trans-acting gene . Repressor binds the operator to repress (turn off) the operon. Catabolite Activator Protein (CAP) binding site  is a  positive  regulatory site located just upstream of the lac operon promoter, where the  catabolite activator protein  ( CAP ) binds.  The CAP is a  dimer  protein, which has binding sites for  cAMP  and DNA. When cAMP binds CAP, its  affinity  for the DNA increases. When bound to DNA, CAP  promotes   transcription  by aiding  RNA polymerase  bind to the  promoter  more efficiently. 12/01/21 Dr.SS ., MRSB (UK)

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14. LAC OPERON The  promoter  is the binding site for RNA polymerase, the enzyme that performs transcription. The  operator  is a negative regulatory site bound by the  lac  repressor protein. The operator overlaps with the promoter, and when the  lac  repressor is bound, RNA polymerase cannot bind to the promoter and start transcription. The  CAP binding site  is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter. 12/01/21 Dr.SS ., MRSB (UK)

15. The  lac  repressor The  lac  repressor is a protein that represses (inhibits) transcription of the  lac  operon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, the  lac  repressor gets in RNA polymerase's way and keeps it from transcribing the operon.  When lactose is not available, the  lac  repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the  lac  repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon. 12/01/21 Dr.SS ., MRSB (UK)

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17. … This change in the  lac  repressor is caused by the small molecule  allolactose , an isomer (rearranged version) of lactose. When lactose is available, some molecules will be converted to allolactose inside the cell. Allolactose binds to the  lac  repressor and makes it change shape so it can no longer bind DNA. Allolactose is an example of an  inducer , a small molecule that triggers expression of a gene or operon. The  lac  operon is considered an  inducible operon  because it is usually turned off (repressed), but can be turned on in the presence of the inducer allolactose. 12/01/21 Dr.SS ., MRSB (UK)

18. Catabolite activator protein (CAP) When lactose is present, the  lac  repressor loses its DNA-binding ability. This clears the way for RNA polymerase to bind to the promoter and transcribe the  lac  operon. That sounds like the end of the story, right? Well...not quite. As it turns out, RNA polymerase alone does not bind very well to the  lac  operon promoter. It might make a few transcripts, but it won't do much more unless it gets extra help from  catabolite activator protein  ( CAP ). CAP binds to a region of DNA just before the  lac  operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription. 12/01/21 Dr.SS ., MRSB (UK)

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20. CAP isn't always active (able to bind DNA). Instead, it's regulated by a small molecule called  cyclic AMP  ( cAMP ). cAMP is a "hunger signal" made by  E. coli  when glucose levels are low. cAMP binds to CAP, changing its shape and making it able to bind DNA and promote transcription. Without cAMP, CAP cannot bind DNA and is inactive. CAP is only active when glucose levels are low (cAMP levels are high). Thus, the  lac  operon can only be transcribed at high levels when glucose is absent. This strategy ensures that bacteria only turn on the  lac  operon and start using lactose after they have used up all of the preferred energy source (glucose). 12/01/21 Dr.SS ., MRSB (UK)

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25. Glucose Lactose CAP binds Repressor binds Level of transcription + - - + No transcription + + - - Low-level transcription - - + + No transcription - + + - Strong transcription Summary of  lac  operon responses 12/01/21 Dr.SS ., MRSB (UK)

26. [email protected] 12/01/21 Dr.SS ., MRSB (UK)

27. QUESTIONS TO THINK Operon Structural genes of lac operon Functional genes of lac operon Mechanism behind presence lactose Mechanism behind presence of glucose 12/01/21 Dr.SS ., MRSB (UK)

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