the lac operon - cusb.ac.in · the operon system the control of protein synthesis has been...

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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

http://www.saburchill.com/IBbiology/bio_hp.html

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.



▪ 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



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




Structure and components of the lac-operon of

E.coli


The control of the lac operon



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



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



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



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



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.



◼ 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



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


◼ 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

the lac operon - cusb.ac.in · the operon system the control of protein synthesis has been...

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