Ennetech by Erasmus and Kinkajou Authors

 

 

Erasmus and Kinkajou share their vision of technologies that will help us on our way.

Genes in Operons: Lac ,Trp

 

 

Turning on and turning off DNA transcription can be very complex indeed.

Even the lac operon in prokaryotic DNA

has extra - operon control modifying factors.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Help us to keep working.

Donate $5-$10

because we really need your support.

Our Sites are run on voluntary donations.

 

Donations
Because we need your help
to survive & keep working

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

v

 

 

KinkajouKinkajou : So tell us more about the function of an operon?
ErasmusErasmus : An operon is a functional unit. It can be present in different forms in viruses, sub-bacterial organisms, bacteria, single celled eukaryotic organisms and multi-celled eukaryotic organisms as well. Operons form a response to the need to coordinate the function of one or more related genes to achieve specific biochemical purposes.

The concept was first developed in 1961 to explain research findings. The genes determining the enzymes of a biochemical pathway were generally in a cluster on the chromosome (Salmonella), ((coordinated repression occurred of a number of enzymes in the pathway (Histidine) bio-synthesis)),  AND mutations existed in regulator and operator parts of the E. coli beta -galactosidase system significantly altering the bacterial response to environmental catabolites.


The theory proposed that:

  • Bacterial (prokaryotic) DNA is organised into operons, each containing one or more genes. These genes have related functions and are controlled or regulate together
  • The operon is the chromosomal unit of transcription for the formation of a molecule of messenger RNA of the same length as the operon (in prokaryotes).
  • Gene expression in the operon is regulated by region called the “operator”, located at one end of the operon.
  • Operons may show repression (a decrease of function), or induction (an increase of function).

 

ErasmusErasmus : The activity of genes is controlled by the cell and the environment.

  • Inducible genes are inactive unless circumstances cause them to be activated (“turned on”).
  • Repressible genes are active unless circumstances cause them to be inactivated (“turned off”).
  • Constitutive gene functions are active continually, with no control exerted. This is generally an abnormal situation.

ErasmusErasmus :It was originally believed that polycistronic transcription existed only in prokaryotic cells such as bacteria. However, since early times a number of examples of dicistronic (two gene) transcription have been found in eukaryotes.
Two forms of processing have been proposed to explain how the “double gene containing mRNA” that is transported to the ribosomes in cytoplasm is translated.

  • Firstly, internal ribosome entry sites (IRES) or some other form of translational re-initiation following the stop codon may be responsible for allowing translation of the downstream gene.
  • Alternately, the initial mRNA transcript is processed by 3' end cleavage and trans-splicing to create a monocistronic mRNA that can be transported to the cytoplasm and translated.

Generally in eukaryotes, strings of adjacent or contiguous genes are not found under the control of a single promoter or operator region. Generally each gene has its own promoter operator region.
Once our mRNA is processed (spliced: regardless of whether we're referring to constitutive or alternative splicing), that mRNA gives rise to a single protein upon translation.

 

ErasmusErasmus :An operon may be made up of 3 basic DNA components:

  • Promoter – a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription.
  • Operator – a segment of DNA that a repressor binds to. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes.
  • Structural genes – the genes that are co-regulated by the operon.

Not always included within the operon, but important in its function is a regulatory gene, a constantly expressed gene which codes for repressor proteins. The regulatory gene does not need to be in, adjacent to, or even near the operon to control it.

Repressors in operons can function by:

  • Negative induction e.g. lack operon
  • Negative repressible e.g. the Trp operon
  • Positive induction: an activator protein binds an inducer, undergoes a conformational change, and then can bind to DNA and activate transcription.
  • Positive repression: an activator protein binds an inhibitor, undergoes a conformational change and is then prevented from binding the DNA, stopping activation and transcription of the system.

 

Sigma factors are specialized bacterial proteins that bind to RNA polymerases and orchestrate transcription initiation. Sigma factors act as mediators of sequence-specific transcription, such that a single sigma factor can be used for transcription of all housekeeping genes or a suite of genes the cell wishes to express in response to some external stimuli such as stress.

KinkajouKinkajou : How did we ever suspect this stuff?

ErasmusErasmus : RECOGNISING POLYCISTRONIC TRANSCRIPTION
Three kinds of observation have led to the discovery of operons:

  • The presence of stable dicistronic mRNAs on Northern blots;
  • A special kind of trans-splicing restricted to use within operons; and
  • The presence of trans-spliced genes in such closely spaced clusters that they almost must be co-transcribed.

KinkajouKinkajou : I would have thought operons really should exist within eukaryotes.
Erasmus: The formation of an operon could well be seen as an evolutionary one-way street. As we’ve mentioned before mutations or breakpoints involving operons would likely inactivate all of the downstream genes, giving them no way to be expressed. Likely to be a lethal situation for a cell.
KinkajouKinkajou : So that’s it?
ErasmusErasmus :The issue gets quite complex.

  • Some gene classes are much more likely to be found within operons. For example, genes which code for mitochondrial proteins and genes which code for DNA operations such as transcription, splicing or translation all have a very strong tendency to be located within operon type structures.
  • Genes involved in RNA degradation are also extremely likely to be found within operons. In fact, for most prokaryotes many of these genes are often found within operons.
  • Another theory, recently is that genes that need to be expressed in the oogenic germ line, a far more likely to be found within operon type structures. Operons are more likely to contain genes that do not need to be regulated at all, but which need to be turned on in all tissues at all times. Hence a single regulator may well work well for a number of genes
  • Genes such as transcription factors, collagens, cytochrome P450 proteins all tend to be excluded from operons. This may reflect the fact that these genes require individual regulation, not one promoter regulating a group of genes.

It cannot be seen that genes within an operon are functionally related. There are many operons where there is no obvious relationship between the genes included and encoded. It may well be for these operons, that the genes are expressed from the same promoter because they all need to be expressed in the oogenic germ line.


Lac Operon Structure Lac Operon Structure

ILLUSTRATION
1: RNA polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA. Top: The gene is essentially turned off. There is no lactose to inhibit the repressor, so the repressor binds to the operator, which obstructs the RNA polymerase from binding to the promoter and making lactase. Bottom: The gene is turned on. Lactose is inhibiting the repressor, allowing the RNA polymerase to bind with the promoter, and express the genes, which synthesize lactase. Eventually, the lactase will digest all of the lactose, until there is none to bind to the repressor. The repressor will then bind to the operator, stopping the manufacture of lactase.

 

KinkajouKinkajou : So tell us about the first operon we discovered the Lac operon.

ErasmusErasmus :The”Lac” operon
The first operon investigated was the lac operon in E. coli in 1959. It contains structural genes encoding the proteins important in lactose metabolism as well as regulatory gene regions. Transcription is a process whereby the DNA of the operon is transcribed into an RNA strand. Translation occurs when the RNA is read and the protein is assembled. This operation can occur at the same time, one part of the strand undergoing translation while transcription of the entire strand is occurring. This operon functions in the classical prokaryotic fashion in that all the proteins are translated from same MRA strand.
Because the lac operon is induced by the presence of lactose, it is described as an inducible system.

Further upstream from the Lac operon is a gene called Lac I which produces a repressor molecule. It is because of the action of this molecule that the Lac operon is by default inactive.

Further upstream, though not directly adjacent, to the lac operon is another gene called lac I. Lac I is constitutively (always) expressed. The gene product of Lac I is a repressor molecule. Because this molecule is always present at some concentration (albeit at a low concentration). It is because of the constitutive expression of this repressor molecule that the lac operon is by default inactive.

 

 Lac Operon Functions 1 Lac Operon Functions 1

Lac Operon Functions D Lac Operon Functions 2

Lac I is the name for the repressor gene;
P is the promoter,
O the operator, and
T the termination sequence.
Lac Operon Functions B
Lac Operon Functions 3


The prokaryotic genome is a circular DNA molecule, and we are viewing just one segment of that circle.  The normal condition for a prokaryotic cell is to have a single copy of its genome in the cell.
Dr AXxxxx:CENSORED "ref ...................." by order of “Frobisher” authorised by “The Commandant”.

Lac operon Functions F Lac operon Functions 4

 

Lac Operon Functions C
Lac Operon Functions 5

 

 

Lac Operon Functions F Lac Operon Functions 6

Lac Operon Functions G Lac Operon Functions 7


ErasmusErasmus : Genetic Control in Prokaryotes
Prokaryotes have two levels of metabolic control

  • Control of protein/enzyme production at DNA transcription/RNA transcription (a slow onset offset action)
  • Regulation of enzyme pathways through mechanisms such as feedback inhibition or through allosteric control mechanisms (a rapidly responsive control system. These regulation pathways are capable of fine tuning if the enzyme system is capable of proportional response to inputs versus simple on- off activity only).

The Lac operon in E. coli
The purpose of the operon is to produce enzymes which digest lactose when lactose is plentiful and glucose is low.
Sugars like lactose are “backup” carbohydrates. The bacterial cell only metabolizes lactose if glucose is low and lactose is plentiful.
The lac operon is an example of an inducible operon - it is normally off, but when a molecule called an inducer (in this case lactose) is present, the operon turns on.

Structure of lactose and the products of its cleavage.

Lactose Molecular Structure Lactose Molecular Structure


There are actually two conditions which must be met if the cell needs these enzymes to be produced. Firstly, lactose must be plentiful. Secondly, glucose must be low.  These criteria   act in a positive and negative fashion to control the function of the operon.

ErasmusErasmus : The operon contains three functional genes

  • LacZ produces B-galactosidase.  This enzyme hydrolyses the bond between the two sugars, glucose and galactose
  • LacY produces permease.  This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose.
  • LacA produces B-galactosidase transacetylase. Assaying this enzyme system to determine its function has proved complex.

ErasmusErasmus : The operon contains a repressor gene as well as other regulatory segments

  • Promoter (P) - aids in RNA polymerase binding
  • Operator (O) - "on/off" switch - binding site for the repressor protein
  • Repressor (lacI) gene: Repressor gene (lacI) - produces repressor protein with two binding sites, one for the operator and one for lactose. The lacI gene coding for the repressor lies nearby the lac operon and is always expressed (constitutive).

The repressor protein prevents transcription when bound to the Operator site; CAP allows transcription when bound to the operator site. This is why catabolites repression is a positive control mechanism and the repressor system is a negative control mechanism.)

Positive Control: By catabolites: Lactose

Operation  - if lactose is present:
The repressor protein has a site for binding with lactose. When lactose is present, it binds to this (allosteric) site on the repressor protein and changes the conformation of the repressor protein. The repressor protein is no longer capable of binding to the operator (DNA site), so the RNA polymerase is uninhibited. Transcription occurs: hence positive control.

Operation - if lactose is absent:
When lactose is absent, the repressor-lactose complex falls apart and the repressor gene produces the repressor protein, which binds to the operator DNA site. This blocks the action of RNA polymerase, thereby inhibiting transcription.
Because lactose (the catabolite) is the activator/ inactivator, the repressor protein is said to be under allosteric control.
(Allosteric control refers to a type of enzyme regulation involving the binding of a non-substrate molecule, known as the allosteric effector, at locations on the enzyme other than the active site. The name "allo" means other and "steric" refers to a position in a certain amount of space.)

 

Operation - If lactose is not present:
Catabolite repression, and involves a protein called catabolite-activating protein (CAP).
CAP has an affinity for the promoter region of the lac operon, and unless CAP is bound to that region, RNA polymerase will not bind to the promoter, and transcription will not occur. (Contrast this to the situation with the lac repressor.

In the Presence of Glucose: Negative Control: Catabolite Repression
Negative control means that an active substance (glucose) acts to turn off the operation of the operon. This promotes energy conservation as it is wasteful to manufacture the enzymes needed to metabolize lactose if there is no lactose available to be used. Under normal conditions the genes for producing the enzymes for metabolizing lactose need to be inactive.

Operation – if glucose is present:
Glucose is the preferred energy source if available. E. coli will preferentially break down glucose over lactose. If glucose is present, the function of the “Lac” operon is repressed
The presence of high levels of glucose in the cell inhibits the activity of adenyl- cyclase, thus reducing the production of cAMP: If the concentration of glucose is high, the concentration of cAMP is low

The regulatory protein is known as CAP, and to be active it must bind cAMP forming a regulatory protein - cAMP receptor protein complex (CRP).
RNA polymerase has a low affinity for the promoter of the lac operon unless helped by CRP. So in the presence of low levels of cAMP (cyclic AMP),
CRP forms at only low levels, and RNA polymerase does not bind to the promoter for the Lac operon.
In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase,

 

ErasmusErasmus : The second control mechanism is a response to glucose,
to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below, about 60 bp upstream of the transcription start site),[3] which assists the RNAP in binding to the DNA

Operation – if glucose is absent:
If the concentrations of glucose is low and lactose is high, the concentration of cAMP will be high, CRP will be activated and bind to the DNA which will promote RNA polymerase binding and initiate transcription

This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases.


 

 

,

ErasmusErasmus : So these three lactose metabolism genes are under dual control.

  • The CAP system prevents lac operon activity when glucose is plentiful because high glucose levels lead to reduced CAP availability, and CAP is necessary for this operon to function.
  • The repressor system prevents lac operon activity when lactose is not available because the repressor protein binds to the Operator site and prevents transcription. The repression can only be removed when lactose is high because allolactose is necessary to inactivate the repressor.
  • The combination of these two control mechanisms ensures that these enzymes will be produced only under conditions in which glucose is low and lactose is high.





However, the lac operon is also under positive control.

  • In the absence of glucose uptake, a protein in the membrane called adenylate cyclase is phosphorylated, leading to the synthesis of a messenger molecule called cyclic AMP (cAMP). cAMP binds to another protein called CRP to form a cAMP-CRP complex which, when bound to DNA, induces lac operon transcription by increasing the affinity of RNA polymerase to the promoter region, in spite of the presence of the repressor molecule.
  • This means that when glucose supplies are depleted and the cell needs to turn to metabolising what lactose is available, it can. This process is called catabolite repression.

ErasmusErasmus : Complications

  • The Lac operon is more complex than this basic summary. Two additional operators have been discovered to be involved in Lac regulation. One lies 92 base pairs upstream of the operator. And the other lies about 401 base pairs downstream of the operator. Dual mutations of these additional operators can depress the function of the operon up to 70 fold.
  • Repressor is bound quite stably to DNA, yet it is released rapidly by addition of inducer. Therefore it seems clear that inducer can also bind to the repressor when the repressor is already bound to DNA. It is still not entirely known what the exact mechanism of binding is.
  • Additionally, there is an abundance of non-specific DNA sequences to which the repressor can bind. Essentially, any sequence that is not the operator, is considered non-specific. Studies have shown, that without the presence of non-specific binding, induction (or un-repression) of the Lac-operon could not occur even with saturated levels of inducer

    It had been demonstrated that, without non-specific binding, the basal level of induction is ten thousand times smaller than observed normally. This is because the non-specific DNA acts as sort of a "sink" for the repressor proteins, distracting them from the operator. The non-specific sequences decrease the amount of available repressor in the cell. This in turn reduces the amount of inducer required to un-repress the system. [6]

ErasmusErasmus : Structure of the lac operon

Lac Operon Strucure in DNA

Lac Operon Functions G

ILLUSTRATION
The lac operon. Top: Repressed, Bottom: Active.
1: RNA Polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA.

Lac Operon Function J

1: RNA Polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA. Top: The gene is essentially turned off. There is no allolactose to inhibit the lac repressor, so the repressor binds tightly to the operator, which obstructs the RNA polymerase from binding to the promoter, resulting in no laczya mRNA transcripts. Bottom: The gene is turned on. Allolactose inhibits the repressor, allowing the RNA polymerase to bind to the promoter and express the genes, resulting in production of LacZYA. Eventually, the enzymes will digest all of the lactose, until there is no allolactose that can bind to the repressor. The repressor will then bind to the operator, stopping the transcription of the LacZYA genes.

 

KinkajouKinkajou : Can give us an example of another operon, that works differently?
ErasmusErasmus : We’ll talk next about the Trp operon.
The Trp operon is an example of a repressible operon - it is normally on but when a molecule called a repressor is present the operon turns off.

Tryptophan Operon E Coli

The Trp operon in E. coli was the first repressible operon to be discovered. This operon controls five structural genes which code for enzymes needed for the production of tryptophan, including the key enzyme tryptophan synthetase. These genes are called Trp E, Trp D, Trp C, Trp B and Trp A.

The tryptophan (Trp) operon in E. coli is a negative control repressible system. The Trp operon contains a promoter which binds to RNA polymerase.  Also within the Trp operon, the repressor gene Trp R produces a protein which binds to the operator along with the amino acid Tryptophan, blocking transcription. The Trp repressor protein is an inactive under normal conditions allowing Trp synthesis when the environmental supply is low. It operates when the amino acid tryptophan is plentiful, reducing excess synthesis. When tryptophan accumulates as an end product of the biosynthetic reaction, tryptophan binds to the inactive repressor protein at an allosteric site. The conformation changes and enables the repressor + tryptophan complex to bind to the operator, repressing the function of operon.

 

It is a negative control because the system produces a repressor which functions to turn off the operon.   Repression occurs when a critical substance (the amino acid tryptophan) is abundant in the cell.

Tryptophan Operon Functions B Tryptophan Operon Functions B

 

Tryptophan Operon Function C
Tryptophan Operon Function C

ErasmusErasmus :
This is an example of feedback control. This occurs when the end product of a process (in this cases, tryptophan) functions to inactivate the process (in this case, to repress the operon).  This allows a consistent steady-state concentration over the level of the key substance to be maintained in the system.