BY:PRIYANKA PATEL Msc.(B.

T) SEM-I ROLL NO:47

Cells must only express genes when needed Gene expression (transcription, translation) takes up large amounts of cellular energy and resources Cells live frugal lifestyles they conserve energy and resources So genes will only be expressed when their products are needed.

Bacteria control genes at the transcriptional level In other words, the gene is either transcribed or not, based on certain external stimuli

1961 /1965

Francois Jacob & Jacques Monod

 first to describe operon system  coined the phrase operon

2005-2006 Jacques Monod

Francois Jacob

An operon is a collection of prokaryotic genes transcribed together on a single mRNA transcript to serve a single purpose Composed of
 An operator, an on-off switch  A promoter  Genes for metabolic enzymes

A Region of DNA that interects with a repressor protein to control the expression of a gene or a group of gene Found within the promoter region or between the promoter and gene encoding regions. On-off switch to gene Controls the access of RNA polymerase + Gene

A DNA sequence at which RNA polymeres may bind ,leading to initiation of transcription Near the operator One promoter control s transcription of all gene in operon Transcribed as 1 unit & a single mRNA is made

It code for the enzymes themselves. The structural genes of an operon usually lies adjacent to one another,and the RNA polymarase moves from one structural gene to the next , transcribing all of the genes into a singal mRNA. This extended mRNA is then translated into the various individual enzymes of the metabolic pathway. Turning on one gene turns on all the producing gene of an operon.

Catabolic operon:An operon composed of genes whose products degrade organic compounds. Biosynthetic operon:An operon composed of genes whose products are involved in synthesizing compounds,such as amino acids or vitamins,rather than degrading them.

Inducer: A signal molecule that, when bind to a regulatory protein, produces an increase in the expression of a given gene Repressor: The protein that binds to the regulatory sequence or operator for a gene,blocking its transcription

Corepressor:The small molecule that associate with an aporepressor to form an active repressor.(e.g.,tryptophan is the corepressor of the trp operon). Aporepressor:A repressor in an inactive form ,without its corepressor.

A repressible operon is one that is usually on; binding of a repressor shuts off transcription The trp operon is a repressible operon An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription The classic example of an inducible operon is the lac operon

Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate CAP helps regulate other operons that encode enzymes used in catabolic pathways

The trp operon contains 5 structural genes ,TRP -E,D,C,A,B. These gene encode for 3 enzymes that turn chorismic acid into Tryptophan. The operon is not expressed when the cell contains sufficient amounts of tryptophan. The operon is expressed when levels of tryptophan are low.
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The trp operon is negatively regulated by the trp repressor protein  trp repressor binds to the operator to block transcription  binding of repressor to the operator requires a corepressor which is tryptophan  low levels of tryptophan prevent the repressor from binding to the operator
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TrpR protein subunits

Tryptophan (corepressor)

DNA

Converts chorismate to anthranilate using ammonia. Anthranilate is an amino acid that is later broken down into another amino acid, Trysoine.

A type of Glycosyltransferase that participates in the synthesis of Tryptophan Produces an amino acid called Anthranilate which ,by the help of TRP E, is broken down into another amino acid, Trysoine.  Converts -(5-phospho-D-ribosyl)anthranilate and diphosphate to anthranilate and 5-phospho-alpha-D-ribose 1-diphosphate. It is 1 of the 2 components that together encode Anthranilate synthetase

Involved in the synthesis of tryptophan and amino acids. It is a type of lyase that cleaves carboncarbon bond.  this ability allows it to converts 1-(2carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate to C(1)-(3-indolyl)-glycerol 3phosphate and carbon dioxide and water

There are two beta subunits which form a dimer Beta dimer catalyzes the formation of Ltryptophan from L-serine and 1-(indol-3yl)glycerol 3-phosphate

Tryptophan synthase is an enzyme found in plants and bacteria There are two separate alpha subunits They catalyze the formation of indole and glyceraldehyde 3-phosphate from indoleglycerol phosphate in tryptophan biosynthesis, which is needed by the B subunits.

Tryptophan present
Regulator Gene Promoter
RNA Polymerase

Operator

Attenuator

Structural Genes

NO TRANSCRIPTION

trpR mRNA Q: Why might the cell want to produce an aporepressor that is only activated by the operon s end product? TrpR aporepressor + corepressor (can bind to operator)

+ tryptophan (corepressor) TrpR protein (homodimer)

Tryptophan Absent
Regulator Gene Promoter
RNA Polymerase

Operator

Attenuator

Structural Genes

TRANSCRIPTION trpR mRNA

TrpR protein (homodimer)

TrpR aporepressor (cannot bind to operator)

The Attenuation Mechanism of trp Operon

Attenuator:A region of DNA upstream from
one or more structural genes, where premature transcription termination(attenuation)can occur.

 Mechanism of Attenuation When Ribosome reaches Trp codon

Trp is scarce Trp is abundant

Attenuation model in Trp starved cells.

Attenuation model in Trp non-starved cells

Generality of Attenuation

The TrpR repressor negatively regulates not only the transcription of the trp operon but also the transcription of its own gene,trpR. In the absence of the TrpR protein,the transcription of the trpR gene is about 5 times higher then in the presence of TrpR. When the product of a gene regulates the expression of its own gene,it is called autoregulation.

L- ara operon was the first e.g. of positive regulation in bacteria to be discovered.Its usually called the ara operon. The ara operon contains 3 structural gene,ara-A,B,D together with the I and O sites. The gene of this operon are responsible for converting L- arabinose into D-xylulos-5phosphate,which can be used by other path ways.

The operon is not expressed when the arabinose is absent in the cell. The operon is expressed when the arabinose is present in the cell.

P t O2 O1 araC
CRP

I1 I2 araB

araA

araD

AraC dimer
Activator/ Repressor

araO1:It is an operator site. AraC binds to this site and represses its own transcription from the PC promoter. In the presence of arabinose, however, AraC bound at this site helps to activate expression of the PBAD promoter. araO2:It is also an operator site. AraC bound at this site can simultaneously bind to the araI site to repress transcription from the PBAD promoter araI :It is also the inducer site. AraC bound at this site can simultaneously bind to the araO2 site to repress transcription from the PBAD promoter. In the presence of arabinose, however, AraC bound at this site helps to activate expression of the PBAD promoter.

CRP binds to the CRP binding site. It does not directly assist RNA polymerase to bind to the promoter in this case. Instead, in the presence of arabinose, it promotes the rearrangement of AraC when arabinose is present from a state in which it represses transcription of the PBAD promoter to one in which it activates transcription of the PBAD promoter.

ara A :Encode L- Arabinose isomerase which converts L- Arabinose into L- Ribulose. ara B :Encode L- Ribulose kinase which phosphorylates L- Ribulose to L-Ribulose-5phosphate. ara D :Encode L-Ribulose-5-phoshate epimerase which converts L-Ribolose-5phosphate into D-Xylulose-5-phosphate. which can then be metabolized via the pentose phosphate pathway.

When arabinose is absent, there is no need to express the structural genes. AraC does this by binding simultaneously to araI and araO2. As a result the intervening DNA is looped. These two events block access to the PBAD promoter which is, in any case, a very weak promoter (unlike the lac promoter):

Arabinose absent

Negative control

Blocked

When arabinose is present, it binds to AraC and allosterically induces it to bind to araI instead araO2. If glucose is also absent, then the presence of CRP bound to its site between araO1 and araI helps to break the DNA loop and also helps AraC to bind to araI:

Arabinose present

Positive control
O2

araC araB O1 CRP araI PC PBAD A D

Transcription

AraC can bind to three sites (araO, araI1, and araI2) with different affinities
(a) No arabinose present: When AraC is bound to araO and to araI1, looping of DNA occurs and prevents transcription

(b) Arabinose present: Arabinose causes allosteric change in AraC so that it cannot bind to araO AraC interacts with RNA polymerase only when both araI1 and araI2 are occupied
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Fig. 15.23

The AraC protein not only regulate the ara operon but also negatively autoregulates its own transcription. If the concentration of AraC becomes too high,its synthesis will again be repressed.

There are several mutations that could prevent the expression of the arabinose operon genes and even cause the cell to die . A mutation in the ara A gene will cause the bacterial cell to become arabinose negative. This means that the bacterium can no longer use arabinose as a carbon source. It will not utilize it if the cell is grown in a minimal media containing arabinose,. A mutation in the ara B gene will also result in the same state. However, a mutation in the ara D gene will result in cell death.

After the ara B gene product degrades Lribulose to L-ribulose-5-phosphate, epimerase is not synthesized and cannot break down Lribulose-5-phosphate which accumulates in the cell. L-ribulose-5-phosphate is toxic to the cell when present in high levels. Mutations could also occur in the ara C gene causing the promoters, PBAD and Pc, to become inactive and the arabinose operon remains permanently repressed.

It is also regulated through catabolite repression , so the gene for arabinose utilization are not expressed if the medium contains a better carbon source. CAP may help open the loop of DNA created when AraC bind to araO2 and ara1.opening the loop may prevent AraC from binding to araO2 and araI1, facilating the binding of AraC to araI1 and araI2 and the activation of transciption from pBAD. Thus, the nt of glucose or another carbon source better then arabinose enhances the transcription of the ara operon.

Scientists modified the arabinose operon in pGLO to express the GFP gene Tag Cells (to detect specific cells) Act as a reporter gene

Source of bioluminesce nce when exposed to UV light

Molecular Biology of the Gene: Watson et al. Molecular Genetics of Bacteria: Synder & Champness www.google.in