the lac operon. in bacteria, the genes involved in the same process are often clustered together....
TRANSCRIPT
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 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.
Lactose catabolism
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
Inactive repressor-effector complex
RNA polymerase
Active repressor binds
to operator
The lac operon
P lacI P lacO lacZ lacY lacA
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
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
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
Inactive repressor-effector complex
RNA polymerase
Active repressor binds
to operator
The lac operon
P lacI P lacO lacZ lacY lacAThis means that there is only one mRNA that encodes three proteins. Each coding region has its own start and
stop codon
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
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 hasIts own promoter and encodes a repressor
protein. It is not part of the operon
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
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
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
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
DNA
LacIrepressor
RNA polymerase
Active repressor binds
to operator
Lactose absent: operon switched off
The lac operon
mRNA
P lacI P lacO lacZ lacY lacA
Transcription
translation
-galactosidase enzyme
lactose permease
lactose trans-
acetylase
DNA
mRNA
protein
(polycistronic message)
lactose
LacIrepressor
Inactive repressor-effector complex
RNA polymerase
P lacI P lacO lacZ lacY lacA
The lac operon
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
The properties of various mutants allowed Jacob and Monod to work out how operons work.
lac mutants
The lac operon
P O lacZ lacY lacA
Active -galactosidase enzyme
DNA
mRNA
protein
DNA
DNAX
XX
mRNA
protein
lacZ mutations are recessive
The lac operon
Lactose catabolism
• 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
DNA
LacIrepressor
RNA polymerase
Active repressor binds to both operators
X
XNo active
repressor to bind to
operator
P lacI P lacO lacZ lacY lacA
P lacI P lacO lacZ lacY lacA
The lac operon
lacI mutations are recessive
• 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
DNA
mRNA
LacIrepressor
RNA polymerase Active repressor binds only
to wild type operator
X DNA
lacOc mutations are dominant
The lac operon
P lacI P lacO lacZ lacY lacA
P lacI P lacO lacZ lacY lacA
• 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 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.
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
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
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.