the lac operon - cusb.ac.in · the operon system the control of protein synthesis has been...
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Central University of South BiharPanchanpur, Gaya, India
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THE lac OPERON and Trp
operon
© 2007 Paul Billiet ODWS
Programme: M.Sc. Biotechnology; 2nd semester
Molecular Biology and Genomics
Course Instructor: Dr. Krishna Prakash
The Operon system◼ The control of protein synthesis has been
elucidated in E.coli.
◼ Wild E.coli growing on minimal medium (glucose+nitrogen source) is able to synthesise all the amino acids need for its growth.
◼ This synthesis requires the presence of certain enzymes, which are produced by the growing E.colicells.
◼ If the required amino acids are supplied in the culture medium, the cells stop synthesising these amino acids and enzymes required for the synthesis of these amino acids are not produced in the E.coli cells.
© 2007 Paul Billiet ODWS
▪ This is called repression, and the enzymes whose synthesis is repressed
are called repressible enzymes.
▪ For example, synthesis of arginine from glutamic acid involves three steps,
each step catalyzed by a separate enzyme. The three steps are:
Glutamic acid OrnithineEnzyme I citrulline
Enzyme II
Enzyme III
arginine
If arginine is absent--------all three enzymes synthesised
If arginine is present---------no one enzyme synthesized
If citrulline is added----------only enzyme III synthesized
• If lactose is present ------------lactose degrading
enzyme synthesis started
• This is referred to as induction and enzymes
produced are known as inducible enzymes
• If lactose is absent then vice-versa step will
take place
• In E.coli this control of gene activity ie.,
repression or induction is operative at the
transcriptional level and needs the presence of
associated genetic controlling elements with
the regulated genes
Operons
◼ An operon is a group of genes that are transcribed at the same time.
◼ They usually control an important biochemical process.
◼ They are only found in prokaryotes.
© NobelPrize.org
Jacob, Monod & Lwoff
© 2007 Paul Billiet ODWS
Structure of operon
Effector molecules bind to repressor and change its ability to interact with operator
The lac Operon
• The lac operon consists of three genes
(lac z, y and a)each involved in processing
the sugar lactose
• One of them is the gene for the enzyme β-
galactosidase
• This enzyme hydrolyses lactose into
glucose and galactose
© 2007 Paul Billiet ODWS
© 2007 Paul Billiet ODWS
Structure and components of the lac-operon of
E.coli
The control of the lac operon
© 2007 Paul Billiet ODWS
Four situations are possible1. When glucose is present and lactose is absent the E.
coli does not produce β-galactosidase.
2. When glucose is present and lactose is present the E. coli does not produce β-galactosidase.
3. When glucose is absent and lactose is absent the E. coli does not produce β-galactosidase.
4. When glucose is absent and lactose is present the E. coli does produce β-galactosidase
© 2007 Paul Billiet ODWS
1. When lactose is absent◼ A repressor protein is continuously synthesised. It sits on
a sequence of DNA just in front of the lac operon, the Operator site
◼ The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing
Regulator
genelac operon
Operator
site
z y aDNA
I O
Repressor
protein
RNA
polymeraseBlocked
© 2007 Paul Billiet ODWS
2. When lactose is present◼ A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at another active site (allosteric site)
◼ This causes the repressor protein to change its shape (aconformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site
z y a
DNA
I O
© 2007 Paul Billiet ODWS
2. When lactose is present◼ A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at another active site (allosteric site)
◼ This causes the repressor protein to change its shape (aconformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site
Promotor site
z y aDNA
I O
© 2007 Paul Billiet ODWS
3. When both glucose and
lactose are present
◼ This explains how the lac operon is
transcribed only when lactose is present.
◼ BUT….. this does not explain why the
operon is not transcribed when both
glucose and lactose are present.
© 2007 Paul Billiet ODWS
◼ When glucose and lactose are present RNA
polymerase can sit on the promoter site but it is
unstable and it keeps falling off
Promotor site
z y aDNA
I O
Repressor protein
removed
RNA polymerase
4. When glucose is absent and
lactose is present◼ Another protein is needed, an activator protein. This
stabilises RNA polymerase.
◼ The activator protein only works when glucose is absent
◼ In this way E. coli only makes enzymes to metabolise other sugars in the absence of glucose
Promotor site
z y a
DNAI O
Transcription
Activator
protein
stabilizes the
RNA
polymerase
© 2007 Paul Billiet ODWS
CAP ( catabolize activator protein)
◼ A regulator gene crp present else where in the E.coli genome produces a
regulator know as CAP.
◼ Also known as CRP (cyclic AMP receptor protein)
◼ CAP protein binds to controlling element near the lac promoter there by
enhancing transcription (positive control)
CAP continued……
◼ The addition of glucose causes CAP protein to leave the controlling
element and lac transcription slows down to very low level.
◼ Presence of glucose inhibits the production of lactose utilization enzymes,
a phenomenon known as catabolite repression.
◼ This repression is brought about by two intermidiates molecules, CAP and
cAMP
◼ In presence of cAMP, CAP stimulates the transcription of lac operon
◼ When glucose is added then the cAMP level goes down (reason unknown)
and this lowers the activity of activator CAP, which results in drop in
transcription of lac operon genes
◼ When glucose and lactose is present in medium , E. coli utilizes it as a
carbon source over lactose.
◼ Thus, CAP function as a activator and cAMP as a effector molecule.
◼ Lac promoter in fact contains 1. RNA pol. Binding site and 2. CAP binding
site.
trp operon
© 2007 Paul Billiet ODWS
◼ The trp operon codes for an enzymes
needed for the synthesis of tryptophan
◼ This is an anabolic pathway as opposed to
the catabolic pathway for the lac operon
◼ Bacterial cells should have the trp operon
“ON” in the absence of tryptophan and
“OFF” in the presence of tryptophan
• The trp operon consists of a promoter, an operator, a leader (trp L), an attenuator,
and 5 structural genes, designated trp E,trp D, trp C, trp B, and trp A.
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Regulation of the E.coli trp operon
• Trp R is a regulatory
homodimer molecule
• Each homodimer contains HTH
motif which interact with half of
the trp operon
• Trytophan function as a
molecular “on” or “off” switch for
trp operon
• Charles Yonofsky in 1972
showed the function of Trp R
• In his experiment he made
E.coli mutants that lack a
functional Trp R protein showed
increase in trp operon
transcription after being starved
for W.
• It means Trp R function as a
regulatory molecule for trp
operon
Reference Book
• Cell Biology By Gerald Karp 7th Edition
• The Cell: A Molecular Approach by Geoffrey
M. Cooper