In addition to the regulatory system described above, there is another mechanism for ensuring optimal substrate metabolism. If glucose is present, bacteria will use this as a substrate of preference, and so the cell has a mechanism for switching off the lac operon, when glucose is available. This mechanism is called catabolite repression. Glucose inhibits adenyl cyclase, an enzyme that catalyzes ATP → cAMP. cAMP binds to the catabolite activator protein (CAP), and bound CAP promotes a higher rate of lac operon transcription. So if glucose levels are high, cAMP levels are low, and CAP doesn’t activate, so transcription rate of the operon is low, and glucose is metabolised instead. Once all glucose has been used up, then cAMP levels begin to rise again, causing activation of the CAP, and hence a step-up for the lac genes expression.
The trp operon
This operon is also found in E.coli, and consists of 5 genes, again transcribed as a single mRNA strand, encoding enzymes involved in the biosynthetic pathway of tryptophan. This operon is under repressible regulation, in that the presence of tryptophan can prevent the transcription of the operon. As with the lac operon, upstream from the genes is a sequence coding for a protein trp repressor, and, in the presence of tryptophan, this repressor can bing to the trp operator, thus inhibiting gene transcription, and preventing unnecessary energy-loss by forming enzymes for the production of amino acids readily available.
This is an example of tryptophan acting as a corepressor with the trp repressor to inhibit its own synthesis by end product inhibition.
Attenuation
There is another mechanism whereby the trp operon may be inhibited from being transcribed without need, and is related to the structure of the operon, and the ability of the DNA to form loops:
Under normal conditions, formation of the 1:2 and 3:4 hairpin loops is energetically favourable.
Attenuation depends on the fact that transcription and translation are tightly coupled in E.coli- translation occurs whilst the gene is still being transcribed.
When tryptophan is in abundance, the ribosome can rapidly translate the trp codons before the operon genes, thus occluding sequence 2 and allowing the 3:4 transcription terminator hairpin loop to form- this attenuates the trp operon.
However, when tryptophan levels are low, the ribosome stalls, as it cannot rapidly translate the pre-operon trp operon. Since the ribosome halts at sequence 1, a 2:3 hairpin loop forms, preventing a 3:4 terminator loop from forming, and thus allowing transcription of the trp operon to continue.
And a final method is by regulation by alternative sigma factors. RNA polymerase is the enzyme responsible for DNA transcription, and in bacteria this enzyme is composed of 5 polypeptide subunits, one of which is called sigma factor (σ). Sigma factor is responsible for initiating transcription by recognizing bacterial promoter DNA sequences. Bacteria such as E.coli retain the ability to make alternative sigma factors, that recognize different sets of promoters- resulting in RNA polymerase transcribing different sets of genes. Since environmental conditions can dictate changes in the type of sigma factor used, this is a way of regulating gene expression.
The most common factor used by E.coli is σ70, although alternative sigma factors may be seen under different conditions, such as:
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heat shock- if E.coli is exposed to high temperatures, it begins to transcribe a set of 17 proteins that are necessary for its continued survival in the harsh environment. The alternative factor σ32 is produced, and this recognizes promoters for the otherwise-not-expressed heat shock genes.
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Sporulation in Bacillus subtilis- B.subtilis undergoes sporulation as a response to adverse environmental conditions. Sporulation involves a major change in the cell’s usual activity, requiring production of specific proteins not usually expressed and shut-down of the protein usually synthesised. The bacteria achieves this through alternative sigma factors.
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Bacteriophage σ factors- phages are viruses that attack bacteria by attaching to the bacterial surface and injecting their DNA core into the host cell, and reproducing. Some phages are able to use the host’s RNA polymerase, and supply it with alternative sigma factors, which means the bacteria transcribes the phage genes preferentially. Other phages produce a series of sigma factors that allow temporal control of their own expression- the genes transcribed earlier by the host polymerase containing a sigma factor that can direct the transcription of later genes.
Eukayotic genes have a more complex pattern of regulation. Not all cells transcribe the same genes- it is the pattern of gene expression that determines the characteristics of a cell and its role in the organinsm. For example, in lymphocytes which produce antibodies to fight infection the genes that encode the polypeptides that make up the antibodies are expressed at a high level, and the pattern of expressed genes may vary during the cell’s lifetime (eg RBCs develop by differentiation from primitive progenitor cells).
The method of regulation favoured by eukaryotes is determining the rate at which genes are transcribed into mRNA. Abundant proteins will be transcribed at very high rates, and rare proteins at much lower rates. The regulation is achieved by the interaction of gene promoters and DNA binding proteins (transcription factors).
Cis-acting regulatory sequences are present in the promoter, these are recognized and bound by transcription factor proteins.
Initiation of gene transcription by RNA polymerase II in eukaryotes occurs by formation of a transcription initiation complex (TIC), consisting of RNA polymerase II and a number of associated proteins known as basal transcription factors binding to the TATA box. The TATA box is a sequnce that lies at the promoter, about 25-30bp upstream of the start of the gene, and locates the RNA polymerase II in the correct position to begin transcription.
It is the additional transcription factors, synthesised in the cytosol, that play a role by binding to other DNA sequences within the promoter (cis-acting elements), to increase or decrease the rate of gene transcription.
There are many different transcription factors, and they each recognize a specific
sequence.
Transcription factors may bind to sequences known as upstream regulatory elements, these may be up to 200bp upstream of the gene sequence. Examples of UREs found within the promoter sequence of many eukaryotic genes are the SP1 box and the CAAT box.
Other UREs are associated with only a few specific genes, and are responsible for limiting the transcription to certain tissues, or in response to certain steroids (steroid hormones control metabolism by entering the target cell, binding to the specific steroid hormone receptor in the cytoplasm, which releases the receptor from an inhibitory protein that normally keeps the receptor within the cytoplasm. The now-free hormone-receptor complex dimerizes and migrates to the nucleus, where it binds to a hormone response element within the gene promoter. There it acts as other transcription factors do, by interacting with the initiation complex to increase transcription rate.
Non-steroid hormones (eg the polypeptides insulin, glucagon, cytokines) do not enter the target cell, rather they bind to cell surface receptors, and may cause transcriptional control via a second messenger signal transduction system. This is a method of transcription regulation separate from transcription factors.
And so hormone response elements are an example of specific UREs, whereas the UREs SP1 box and CAAT box are ubiquitous.
Transcription factors may also bind to sequence elements known as enhancers, and these are found at long distances from the gene.
It was originally thought that enhancers were separate to promoter-UREs, because they:
- can activate transcription over long distances
- can be found upstream/downstream of the gene being controlled
- are active in any orientation with respect to the gene
But it is now accepted that enhancers and promoter-UREs show strong similarities physically and functionally.
So how do transcription factors interact with the DNA?
Well, transcription factors consist of 2 domains- a DNA-binding domain, and an activation domain, and some transcription factors operate as dimers, and so have dimerization domains.
DNA-binding domains
There are 3 main types of binding domain:
* HELIX-TURN-HELIX – 2 alpha-helices separated by a beta-turn. The recognition helix binds to the DNA by making contact through the major groove
* ZINC FINGERS- 2 beta-strands and an alpha-helix that also makes contact through the major groove.
* BASIC DOMAINS- found in some transcription factors- ususally with leucine zipper or helix-loop-helix.
Dimerization domains
There are 2 types:
* LEUCINE ZIPPERS-
* HELIX-LOOP-HELIX- 2 alpha-helices separated by a nonhelical loop. The helix-loop-helix dimerization domain is found in the MyoD family of transcription factors that regulate gene expression in developing muscl cells.
Activation domains
There are no motifs that characterize activation domains, although they can be divided into 3 types:
* those containing a lot of acidic amino acids
* those containing a lot of glutamine
* those containing a lot of proline