Transcript lac
The lac operon Lactose catabolism • 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 • 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 • These genes are controlled. E. coli is a successful competitor in the gut because it doesn’t 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 • 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. The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Transcription mRNA LacI repressor lactose Active repressor binds to operator Inactive repressoreffector complex (polycistronic message) translation -galactosidase enzyme protein lactose permease lactose transacetylase The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Transcription mRNA (polycistronic message) translation lie side three Active coding sequences The repressor binds by LacI to operator protein side but there is only one promoter repressor lactose Inactive repressoreffector complex -galactosidase enzyme lactose permease lactose transacetylase The lac operon This means that there is only one mRNA that encodes P lacI P lacO lacZ lacY lacA three proteins. Each coding region has its own start and DNA stop codon RNA Transcription polymerase mRNA LacI repressor lactose Active repressor binds to operator Inactive repressoreffector complex (polycistronic message) translation -galactosidase enzyme protein lactose permease lactose transacetylase P lacI P lacO RNA polymerase LacI repressor lactose operon The separateThe lacIlac gene is not controlled. It has lacZ lacY lacA Its own promoter and encodes a repressor DNA protein. Transcription It is not part of the operon mRNA Active repressor binds to operator Inactive repressoreffector complex (polycistronic message) translation -galactosidase enzyme protein lactose permease lactose transacetylase P lacI RNA polymerase LacI repressor lactose InThe the absence of lac operon lactose, the repressor protein P lacO lacZ lacY binds lacA to a special site in the DNA operon called the Transcription OPERATOR and mRNA prevents RNA (polycistronic message) polymerase from Active translation repressor binds moving along the to operator protein DNA Inactive repressoreffector complex -galactosidase enzyme lactose permease lactose transacetylase The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Transcription mRNA LacI repressor lactose Active repressor binds to operator Inactive repressoreffector complex (polycistronic message) translation -galactosidase enzyme protein lactose permease lactose transacetylase The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Lactose absent: operon switched off mRNA LacI repressor Active repressor binds to operator The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Transcription mRNA (polycistronic message) translation LacI repressor lactose protein Inactive repressoreffector complex -galactosidase enzyme lactose permease lactose transacetylase The lac operon Jacob and Monod Jacob François Jacob and Jacques Monod worked out how the lac operon functioned and they formulated the operon hypothesis. Monod The lac operon lac mutants The properties of various mutants allowed Jacob and Monod to work out how operons work. The lac operon P O lacZ lacY lacA DNA mRNA Active -galactosidase enzyme lacZ mutations are recessive protein Lactose catabolism X DNA X X mRNA protein The lac operon Constitutive mutants • 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. The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Active repressor binds to both operators LacI repressor P lacI P lacO lacZ lacY lacA X No active repressor to X bind to operator lacI mutations are recessive The lac operon A testable prediction • 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. The lac operon P lacI P lacO lacZ lacY lacA DNA RNA polymerase Active repressor binds only to wild type operator LacI repressor P lacI P lacO lacZ lacY lacA X DNA mRNA lacOc mutations are dominant The lac operon • 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 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). The lac operon Catabolic repression The lac operon is also under the control of a positive regulatory protein. E. coli’s 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. 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. The lac operon P lacO lacZ lacY lacA CAP DNA RNA polymerase Transcription mRNA (polycistronic message) translation protein Glucose cAMP -galactosidase enzyme lactose permease lactose transacetylase The lac operon Summary 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) Complementation • Diploid, Haploid • Dominant, Recessive • Homozygous, Heterozygous • Cistron • Cross-feeding Colinearity of the gene and protein Protein structure Haemoglobin Genetic code amino acid sequence The Genetic code Codon Dictionary of the genetic code How the code was deciphered How the code works tRNA and Translation RNA translation (5' 3') Ribosomes Structure of tRNA Anticodon Mechanism of translation Wobble hypothesis Inosine (I) is a rare base found in tRNA, often in the anticodon, capable of binding to adenine, uracil or cytosine. RNA Translation RNA growth (5' 3') RNA polymerase, structure and properties Promoter, consensus Mechanism of translation Termination Suggested reading Regulation of gene transcription (2000) In: An Introduction to Genetic Analysis. pp 336-344. Griffiths, A. J. F,. Miller, J. H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W. M. (Eds). Freeman and Company, New York.