How do genes switch on and off in response to the environment?

[dpsguy]

I wanted to know how genes switch on and off. I understand it is in response to the external environment of the organism and involves something called trancription factors, but I am not very sure of the mechanism. I have studied about operons but it seems to be a rather simplistic concept, not applicable to higher organisms like humans. Could someone help me out?

 

[Monique]

RNA polymerase needs to bind to the start of a gene, for it to be able to transcribe and thus express a gene. Transcription factors are proteins expressed in a cell that have the ability to bind to certain DNA sequences. These DNA sequences lie upstream of a gene in the promotor region. After binding the transcription factors recruit many more proteins and thereby create a complex, this complex can modulate the structure of DNA so that the RNA polymerase transcription complex can bind and start transcribing the gene.

I hope that answers your question, after transcribing the gene you've got messenger RNA (mRNA), which still needs to be processed in several steps in order to get to proteins.

 

[iansmith]

Just to add a few comments.

Some transcription factors are often actively expressed but their action is either inhibited by other proteins or by their phosphorylation state. The switch of off to on is control by other factors such as cell receptor and their associated proteins. The whole process is called signal transduction. This can be found in both bacteria, archea and eukaryotes but the systems in bacteria and archea usually have less components in the system. However, the system can be as complicated and several system can overlap in functions.

http://www.sigmaaldrich.com/img/assets/6460/sig_trans_proreg.gif
http://en.wikipedia.org/wiki/Signal_transduction

In a nutshell, you get a receptor on the surface of the cell that sense an environmental. The environmental signal induce a change of state for the receptor and that change start a cascade of changes downstream of the receptor.

 

[dpsguy]

DNA polymerase needs to bind to the start of a gene, for it to be able to transcribe and thus express a gene. Transcription factors are proteins expressed in a cell that have the ability to bind to certain DNA sequences. These DNA sequences lie upstream of a gene in the promotor region. After binding the transcription factors recruit many more proteins and thereby create a complex, this complex can modulate the structure of DNA so that the RNA polymerase transcription complex can bind and start transcribing the gene.

I hope that answers your question, after transcribing the gene you've got messenger RNA (mRNA), which still needs to be processed in several steps in order to get to proteins.

Thanks Monique!
BTW, shouldn't that be RNA polymerase in the first line?

Just a little problem: if transcription factors(TFs) are protiens, then there must be a regulatory mechanism for them too. Does that mean that there are TFs that regulate TFs? If so, does a feedback mechanism operate, like in operons? And how does the external environment of the cell come into the picture?
The link very helpfully given by Iansmith talks about external stimuli affecting cellular receptors which "can activate many downstream effector proteins". Then does this process bypass nuclear transcription? If not, what role do TFs have to play in this process?

 

[Monique]

Thanks Monique!
BTW, shouldn't that be RNA polymerase in the first line?

You are absolutely right.

Just a little problem: if transcription factors(TFs) are protiens, then there must be a regulatory mechanism for them too. Does that mean that there are TFs that regulate TFs?

Yes, but you wouldn't call them transcription factors, but regulatory proteins. A very well-known and much-used example for gene-regulation is the LacZ gene (part of the lac operon). It is often cloned into a vector and used as a reporter gene.

The way it works is that the lac repressor protein (lacI) binds to the lac operon and thereby prevents transcription. When IPTG is added, the lac operon is de-repressed, due to the binding of IPTG to lacI, preventing it from binding the lac operon. Now the gene can be transcribed and translated into beta-galactosidase, a protein with enzymatic activity. The substrate for the enzyme is X-gal, which when cleaved leaves a water-insoluble blue product, which can be used as a reporter.

A way to envision the use of this LacZ protein is for instance to monitor whether you have cloned (inserted) a gene into a vector (a circular piece of DNA from viral origin). When the system is intact, you will get blue bacterial colonies (these are the 'test tubes' in which you perform the reaction), when the LacZ gene is disrupted by an inserted protein, you get white bacterial colonies.

If so, does a feedback mechanism operate, like in operons? And how does the external environment of the cell come into the picture?
The link very helpfully given by Iansmith talks about external stimuli affecting cellular receptors which "can activate many downstream effector proteins". Then does this process bypass nuclear transcription? If not, what role do TFs have to play in this process?

There are many different pathways known that play a central role in signal transduction from the outside to the inside of the cell. Everything is highly regulated and there are often feedback mechanisms.

A well-known signal transduction pathway is the MAPK pathway, which is a kinase cascade where kinases phosphorylate kinases, which in turn will phosphorylate kinases. In this way the signal is greatly enhanced.

http://www.biocarta.com/pathfiles/h_mapkPathway.asp

The pathway in outline works like this: the epidermal growth factor receptor (EGFR) binds and is activated by the extracellular epidermal growth factor (EGF), by forming a dimer. The receptor-dimer can then cross-phosphorylate the tyrosines on its cytoplasmic tail.

Docking proteins such as GRB2, which contain an SH2 domain, can then bind to the phosphotyrosines. Then a guanine nucleotide exchange factor (GEF) binds to the SH3 domain of GRB2 and becomes active.

The GEF exchanges the GDP for a GTP on Ras, thereby activating it, which can continue to activate the protein kinase Raf. This sets off a whole set of kinases, which in the end go into the nucleus and signal to transcription factors (on the bottom in the diagram: Elk-1 c-FOS c-JUN ATF-2 SP-1 STAT-1 etc).

As seen in the LacZ example, some proteins will promote transcription, others will repress it. There is a whole other mechanism for gene repression/activation, which is based on histone-tail modifications. This is a very complex subject by itself.

 

[dpsguy]

Thanks again for your reply.

Yes, but you wouldn't call them transcription factors, but regulatory proteins. A very well-known and much-used example for gene-regulation is the LacZ gene (part of the lac operon). It is often cloned into a vector and used as a reporter gene.

The way it works is that the lac repressor protein (lacI) binds to the lac operon and thereby prevents transcription. When IPTG is added, the lac operon is de-repressed, due to the binding of IPTG to lacI, preventing it from binding the lac operon. Now the gene can be transcribed and translated into beta-galactosidase, a protein with enzymatic activity. The substrate for the enzyme is X-gal, which when cleaved leaves a water-insoluble blue product, which can be used as a reporter.

Does this mean that regulatory genes like the lacI gene are always being transcribed, irrespective of whether the LacZ gene is active or not?

A way to envision the use of this LacZ protein is for instance to monitor whether you have cloned (inserted) a gene into a vector (a circular piece of DNA from viral origin). When the system is intact, you will get blue bacterial colonies (these are the 'test tubes' in which you perform the reaction), when the LacZ gene is disrupted by an inserted protein, you get white bacterial colonies.

But this will not always work. Even if the gene is inserted properly but the protien does not interfere with the lacI protien,you will still get blue colonies.
Or am I wrong in my understanding?

There are many different pathways known that play a central role in signal transduction from the outside to the inside of the cell. Everything is highly regulated and there are often feedback mechanisms.

A well-known signal transduction pathway is the MAPK pathway, which is a kinase cascade where kinases phosphorylate kinases, which in turn will phosphorylate kinases. In this way the signal is greatly enhanced.

http://www.biocarta.com/pathfiles/h_mapkPathway.asp

The pathway in outline works like this: the epidermal growth factor receptor (EGFR) binds and is activated by the extracellular epidermal growth factor (EGF), by forming a dimer. The receptor-dimer can then cross-phosphorylate the tyrosines on its cytoplasmic tail.

Docking proteins such as GRB2, which contain an SH2 domain, can then bind to the phosphotyrosines. Then a guanine nucleotide exchange factor (GEF) binds to the SH3 domain of GRB2 and becomes active.

The GEF exchanges the GDP for a GTP on Ras, thereby activating it, which can continue to activate the protein kinase Raf. This sets off a whole set of kinases, which in the end go into the nucleus and signal to transcription factors (on the bottom in the diagram: Elk-1 c-FOS c-JUN ATF-2 SP-1 STAT-1 etc).

As seen in the LacZ example, some proteins will promote transcription, others will repress it. There is a whole other mechanism for gene repression/activation, which is based on histone-tail modifications. This is a very complex subject by itself.

Are the TFs being finally signalled here already present in the nucleus? If so, when were they transcribed? Or are the genes which code for these TFs activated at the end of the cascade? In that case, how are they repressd again?

 

[iansmith]

Does this mean that regulatory genes like the lacI gene are always being transcribed, irrespective of whether the LacZ gene is active or not?

In the case of lacI, the gene is always transcribed but it's usually at very low level. LacI transcription is constitutive.

The activity of LacZ is only indicative of the repression of lacZ by LacI.

But this will not always work. Even if the gene is inserted properly but the protien does not interfere with the lacI protien,you will still get blue colonies. Or am I wrong in my understanding?

In this case the structural gene lacZ is being disrupted but lacI is untouched. However, when testing the insertion, lactose or IPTG is added to make sure lacZ would not be repressed by LacI

Are the TFs being finally signalled here already present in the nucleus? If so, when were they transcribed? Or are the genes which code for these TFs activated at the end of the cascade? In that case, how are they repressd again?

For TF, some will be located in the nucleus and some will be located in the cytoplasm. The TF are often constitutively expressed but the activity is regulated by TF-protein interaction. Therefore, regulation of activity is not done of the gene level but rather at the protein level.

Things can get complicated with regulators. It's often a case to case basis and it's usually not a linear regulation. For example, at the end of cascade, some TF might be upregulated leads to the accumulation of TF and thus it reinforces the signal. However, the opposite can be seen in other situation.

 

[dpsguy]

Thanks iansmith!

The functioning of operons seems to be much simpler than, say, the MAPK pathway. Are operons found only in lower organisms like prokaryotes, or also in humans? Conversely, are transcription factors and mechanisms like the MAPK pathway found in lower organisms too?

 

[iansmith]

The functioning of operons seems to be much simpler than, say, the MAPK pathway.

It's all relative to task that each pathway must do. lacI only controls one operon, MAPK controls several pathways. It's all about fine tuning. An all or nothing response requires less check points than something that need a small, medium or a high response. The lacZ is more a like of all or nothing response.

Are operons found only in lower organisms like prokaryotes, or also in humans?

Operon are predominately found in bacteria but, if i remember correctly, there's a few example of operon-like structure found in human and other eukaryotes.

Conversely, are transcription factors and mechanisms like the MAPK pathway found in lower organisms too?

By definition, a TF is a protein that helps RNA polymerase to bind to DNA and initiate transcription. Therefore, TF are technically ubiquitous. However, the term is only use to referred to specific protein found in eukaryotes.

In bacteria, there is proteins that will increase the affinity of RNA polymerase for a certain DNA region and help initiate transcription. These proteins are called sigma factors. Sigma factors play an important role in regulation. A bacteria can have several sigma factors. some sigma factors are also regulated by a membrane bound anti-sigma factor. This anti-sigma binds to the sigma factor which cannot initiate transcription. The sigma factor is release when the anti-sigma senses the signal. The signal is often sense by other protein and for some anti-sigma, proteins will cleave the anti-sigma in order to release the sigma factor.

Similar proteins are found in bacteria but the system is much more simpler. In bacteria, those system are often referred to as two-component regulatory systems. Basically, it's a an histidine kinase membrane receptor and cytoplasmic response regulator. The membrane receptor is autophosphosrylated when the signal is present and the receptor passes the "message" to the response regulator which acts on its target. either DNA or other proteins. Other protein can also have an accessory role or play a crucial role.

 

[dpsguy]

RNA polymerase needs to bind to the start of a gene, for it to be able to transcribe and thus express a gene. Transcription factors are proteins expressed in a cell that have the ability to bind to certain DNA sequences. These DNA sequences lie upstream of a gene in the promotor region. After binding the transcription factors recruit many more proteins and thereby create a complex, this complex can modulate the structure of DNA so that the RNA polymerase transcription complex can bind and start transcribing the gene.

From Wikipedia:

E.coli sigma factors:

σ70 (RpoD) - the "housekeeping" sigma factor, transcribes most genes in growing cells.
σ38 (RpoS) - the starvation/stationary phase sigma factor
σ28 (RpoF) - the flagellar sigma factor
σ32 (RpoH) - the heat shock sigma Factor
σ24 (RpoE) - the extracytoplasmic stress sigma factor
σ54 (RpoN) - the nitrogen-limitation sigma factor
σ19 (FecI) - the ferric citrate sigma factor

According to this, there are different sigma factors for different types of genes. What differentiates these sigma factors and genes from each other? In other words, what is special about, say, nitrogen limiting genes and the corresponding sigma factor? Why can't a flageller sigma factor activate housekeeping genes, since it has to bind to the promoter site, which usually consists of a Pribnow box in most prokaryotes?

Is the same thing found in eukaryotes too, ie. are there different TFs for different types of genes?

 

[Monique]

Transcription factors bind to specific DNA sequences, so they activate specifically the genes that have the DNA sequence in its promoter. A single transcription factor usually regulates several genes in the same pathway, this is ofcourse efficient.

 

[dpsguy]

Transcription factors bind to specific DNA sequences, so they activate specifically the genes that have the DNA sequence in its promoter. A single transcription factor usually regulates several genes in the same pathway, this is ofcourse efficient.

Does this mean that we can predict the function of unknown genes just by looking at the promoter region before it? Do various classes of genes have the same promoter in all species?

 

[Monique]

We are very poor at bioinformatically predicting the binding of transcription factors to promoter regions, when you do that you will find hunderds of transcription factors binding. Usually you need a matrix of TFs that bind together in a group and thereby exert a function. I am not aware of literature where they have compared promoters to find genes in a common pathway, theoretically this is possible. Genomatix is software that could be used for that purpose: http://www.genomatix.de/products/index.html

You must realiaze that promoters are very poorly defined regions, gene-regulatory elements usually lie in a region of 1000 bases upstream of a gene, but can extend beyond that.

 

[dpsguy]

Thanks Monique! You have been very helpful.