Embedded Files
This lecture covers
- What is bacterial expression and its regulation?
- Why regulation is needed for bacterial systems?
- What are different types of regulations and their functionality?
Over-view of gene expression
Over-view of gene expression
The information that are stored in the DNA should be transcribed into mRNA and further translated into proteins. This process collectively referred as gene expression and this functional unit of DNA is referred as gene. As discussed in the previous chapter, a single gene or two and more genes can be transcribed together as single mRNA (monocistronic and polycistronic) and later translated into several proteins.
Hence, a “gene” should have a region where the RNA polymerase binds to initiate the transcription (known as promoter) followed by the gene (or genes) coding for a protein (hereafter referred as structural genes / transcription units / open reading frames) and ends with a sequence, which terminates the transcription (Terminator). Apart from the structural genes region, mRNA also include some additional head and tails. Head region includes a untranslated region (In diagram, as 5`UTR) followed by ribosomal binding site follwoed by start codon. After the stop codon, again UTR (known as 3’UTR) followed by terminator loop will be like a tail. The ribosome binds at ribosomal binding site at head region of mRNA and initates the translation. In bacteria, all the proteins will strat with N formyl methionine (while in eukaryotes, it was methionine).
Summary on Gene Expression
- Transcription of DNA is carried out by RNA polymerase, which adds one ribonucleotide at a time to the 3’ end of a growing RNA chain. The sequence of the template DNA strand determines the order in which ribonucleotides are polymerized to form an RNA chain.
- During transcription initiation, RNA polymerase binds to a specific site in DNA (the promoter), locally melts the double-stranded DNA to reveal the unpaired template strand, and polymerizes the first two nucleotides.
- During strand elongation, RNA polymerase moves along the DNA, melting sequential segments of the DNA and adding nucleotides to the growing RNA strand.
- When RNA polymerase reaches a termination sequence in the DNA, the enzyme stops transcription, leading to release of the completed RNA and dissociation of the enzyme from the template DNA.
- In prokaryotic DNA, several protein-coding genes commonly are clustered into a functional region, an operon, which is transcribed from a single promoter into one mRNA encoding multiple proteins with related functions. Translation of a bacterial mRNA can begin before synthesis of the mRNA is complete.
- In eukaryotic DNA, each protein-coding gene is transcribed from its own promoter. The initial primary transcript very often contains noncoding regions (introns) interspersed among coding regions (exons).
- Eukaryotic primary transcripts must undergo RNA processing to yield functional RNAs. During processing, the ends of nearly all primary transcripts from protein-coding genes are modified by addition of a 5’ cap and 3’ poly(A) tail. Transcripts from genes containing introns undergo splicing, the removal of the introns and joining of the exons. (For more details, please view previous lecture)
What is regulation and why is essential for bacteria?
What is regulation and why is essential for bacteria?
Some enzymes/proteins/RNAs are needed for a bacterium at same levels in all the growth conditions. The expression of these genes will not be controlled and hence referred as constitutive expression. However, most of the macromolecules (proteins/enzymes) are needed only under some condition, not other conditions. For, example, enzymes to utilize lactose sugar are needed only if lactose is available in the medium. If lactose is not present, then this gene need not be expressed. These kinds of genes are said to be under Regulation.
The bacterial system is a single cell doing all the functions with available energy. The metabolic pathways need high energy. To conserve their energy, they switch off or kept at low levels of their enzyme production which they are not needed for a particular condition. Example, if lactose is not present, the lactose utilizing enzymes will be at very low level in their cells. Thus, regulation is a major process in all cells and helps to conserve energy and resources.
Regulation – An over view
For regulating a gene, apart from the components of a gene, few other genes and their proteins are essential.
- Operator: A segment of DNA close to promoter (downstream of promoter) in which regulatory proteins bind and control the expression.
- Regulatory protein: The protein produced by the regulatory gene which attach to the operator and thereby regulate the gene expression. The binding at operator may either initiate the expression or sometimes inhibit the expression.
- Regulatory gene: It is a structural gene, which produce a regulatory protein and thereby regulate the gene’s expression. It is not necessary that the regulatory gene should be near the genes under its control.
Operon
The repressor protein when active can bind to a specific region of the DNA near the promoter of the gene, known as the operator. This region gave its name to the operon, a cluster of genes arranged in a linear and consecutive fashion whose expression is under the control of a single operator.
The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be both co-transcribed and co-regulated to define an operon.
Originally, operons were thought to exist solely in prokaryotes, but since the discovery of the first operons in eukaryotes in the early 1990s, more evidence has arisen to suggest they are more common than previously assumed.
Historical Perspectives
The term "operon" was first proposed in a short paper in the Proceedings of the French Academy of Science in 1960. From this paper, the so-called general theory of the operon was developed. This theory suggested that all genes are controlled by means of operons through a single feedback regulatory mechanism– repression. Later, it was discovered that the regulation of genes is a much more complicated process. Indeed, it is not possible to talk of a general regulatory mechanism, because different operons have different mechanisms. Despite modifications, the development of the concept is considered a landmark event in the history of molecular biology. The first operon to be described was the lac operon in E. coli. The 1965 Nobel Prize in Physiology and Medicine was awarded to François Jacob, André Michel Lwoff and Jacques Monod for their discoveries concerning the operon and virus synthesis. [More details are in lecture 2]
Types of regulation
Types of regulation
Based on the functions of regulatory proteins, the regulatory mechanisms of bacteria are of two types viz., negative and positive regulation.
Negative regulation: It involves the binding of regulatory protein in the operator, to prevent or block the transcription. (Since, the regulatory proteins repress (block) the expression in negatively regulated operons, they are also referred as Repressor Proteins).
Positive regulation: The regulatory protein needs to bind the DNA (not necessary to be at operator) to initiate the expression (in other words, transcription), then it is said to be positive regulation and the operon is positively regulated operon. (Since the regulatory proteins activate the operon’s expression, they are also referred as activator proteins in positive regulatory operons).
Based on the activation of regulatory protein, again the operons can be of two types viz., inducible and repressive operons.
Inducible operons:
Inducible operons:
A regulatory protein is normally bound to the operator, which prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to the regulatory protein and changes its conformation so that it is unable to bind to the operator. This allows for expression of the operon. Such operons are referred as inducible operons.
Inducer: It is a small molecule which binds with repressor protein and thereby initiate the transcription (See the above figure). Later, the enzymes synthesized from the operon will catabolize the inducer. Example: Lactose is an inducer for lac operon, which will initiate the expression.
Repressible operons
Repressible operons
transcription of the operon normally takes place. Repressor proteins are produced by a regulator gene, but they are unable to bind to the operator in their normal conformation. However, certain molecules called co-repressors are bound by the regulatory protein, causing a conformational change to the active site. The activated repressor protein binds to the operator and prevents transcription. Such operons are referred as repressive operons.
Co-repressor: A small molecule, binds with the inactive repressor protein and make it active and block (repress) the transcription (See above figure). The co-repressor will be the product of the enzymes of that operon. Example: Arginine is a co-repressor blocks the arg operon, when sufficient amount of arginine is produced.
When combining the function and activation of regulatory proteins, the operons can be of four types:
- Negative inducible
- Negative Repressible
- Positive Inducible
- Positive Repressible
Negative Regulation
Negative Regulation
A. Inducible Operon (Ex: lac operon)
Lac operon is responsible for the utilization of lactose by E. coli. There are three structural genes essentially needed for the utilization of lactose viz., lacZ encodes for βgalactosidase, responsible for cleavage of the β1,4-glycosidic linkage in lactose resulting in the monosaccharides, galactose and glucose; lacY responsible for lactose permiase, facilitates the transport of lactose to the cells; lacA codes lactose transacetylase, responsible for transacetylation of βgalactosides. All these three enzymes are having common promotor (Plac) and transcribed a single mRNA for all three proteins. Hence, it is a polycistronic.
Apart from these, lacI located upstream of the lac genes is the regulatory gene controlling the lac operon expressions. The LacI is the regulatory protein (also called as Repressor protein), produced by lacI binds with operator (Olac) of lac operon (which is close to promoter; See the figure below) and blocks the expression.
Under normal condition (lactose is not present in the medium), LacI binds with Olac hence no expression of lac gene will takes place. However, when lactose concentration is increased in the medium, the few copies of LacY protein (responsible for transport of lactose) mediate the lactose transport to the cells. The lactose (here as inducer) binds with LacI (repressor protein) and confirmationally changed its active binding site, hence the LacI could not bind with Olac. This facilitates to RNA polymerase to bind and initiate the transcription of polycistronic mRNA. The enzymes will catabolize the lactose to get their energy. Once the lactose concentration is decreased, the LacI protein will again binds with Olac and repress the gene expression.
lac operon induced by lactose by binding with LacI protein (repressor protein)
There are some alalogs of lactose, IPTG (Isopropyl thiogalactoside) whose presence in the cells can binds the LacI protein and “turn on” the operon, but the enzyme β-galactosidase could not metabolize this substrate. Hence it can act as inducer only but not as substrate for the enzyme, hence, referred as Gratuitous Inducer.
Another substrate, Xgal, which cannot act as inducer, but the enzyme (β-galactosidase) can catabolize the substrate. Hence, it acts as substrate for the enzyme but not as inducer. These two chemicals are highly useful to study the regulation of lac operon under lab condition.
Lactose Analogs:
X-gal is converted from a colorless compound to a dark blue compound when it is cleaved by β-galactosidase. ONPG is converted from a colorless compound to a yellow compound. IPTG cannot be cleaved by β-galactosidase but it can bind to LacI and induce the lac operon.
Animation showing lac operon functioning (Youtube)
B. Repressive Operon (Ex. trp operon)
Transcription of most E. coli genes is regulated by processes similar to those described for the lac operon. The general mechanism involves a specific repressor that binds to the operator region of a gene or operon, thereby blocking transcription initiation. A small molecule (or molecules), called an inducer, binds to the repressor, controlling its DNA-binding activity and consequently the rate of transcription as appropriate for the needs of the cell. In this operon, the substrate (lactose), which is going to be catabolized by the enzyme acts as inducer. In contrast to this, in some cases, the product (a synthesizing molecule from the enzyme of the operon) also negatively regulates the operon.
For example, when the tryptophan concentration in the medium and cytosol is high, the cell does not synthesize the several enzymes encoded in the trp operon. Binding of tryptophan to the trp repressor causes a conformational change that allows the protein to bind to the trp operator, thereby repressing expression of the enzymes that synthesize tryptophan. Conversely, when the tryptophan concentration in the medium and cytosol is low, tryptophan dissociates from the trp repressor, causing a conformational change in the protein that causes it to dissociate from the trp operator, allowing transcription of the trp operon. In this case, tryptophan is referred as Co-repressor, and the operon is said to be Repressive Operon.
The biosynthesis of the aromatic amino acid tryptophan requires the activity of three enzymes, anthranilate synthetase (trpE) complexed with phosphoribosylanthranilate transferase (trpD), phosphoribosyl-anthranilate isomerase (trpC), and tryptophan synthetase (trpA and trpB) to convert chorismate to tryptophan. The five trp structural genes encoding the three tryptophan biosynthetic enzymes are physically linked and coordinately regulated as an operon. The trp genes are arranged in the order that their encoded enzymes function in the biosynthetic pathway. The goal of a biosynthetic or anabolic pathway is to synthesize the end product only when it is needed. The trp operon is repressed by TrpR, a transcriptional repressor, when levels of tryptophan are high in the cell. The gene encoding the TrpR repressor is not located near the trp operon. Even though expression of both the lac and trp operons are regulated by repression (in other words Negatively regulated), the mechanism by which the LacI and TrpR repressors work is very different. The LacI repressor when bound to its effector molecule (i.e. lactose) cannot repress transcription. In contrast, the TrpR repressor must be bound by an effector molecule, in this case tryptophan (referred as Co-repressor), for it to repress transcription.
Animation of try operon functioning (youtube)
Positive Regulation
Positive Regulation
Either glucose alone or lactose alone used as carbon source, E. coli will have its growth curve as described elsewhere. If glucose alone, undergo glycolysis and energy will be produced and if lactose alone, with the help of inducible lac operon, lactose will be catabolized and energy will be produced. In contrast to these, When glucose and lactose are present in the medium, E. coli will not utilize lactose until the glucose is utilized. As soon the glucose is exhausted, then the lactose utilization will be initiated. Here, the glucose regulates the lac operon.
How glucose regulates the lac operon?
If lactose and glucose are added to a culture of wild-type E. coli cells, the lac operon is not induced. No lac mRNA or gene products are made. This effect of glucose is the result of a second regulatory mechanism, catabolite repression. Catabolite repression affects not only lac gene expression but also other operons that catabolize specific sugars such as galactose, arabinose, and maltose. Contrary to its name, catabolite repression describes an activating mechanism, involving a complex between cAMP (cyclic adenosine monophosphate) and the catabolite activator protein (CAP; also called cyclic AMP receptor protein or CRP).
Cyclic AMP is a key molecule in many metabolic control systems, both in prokaryotes and eukaryotes. Because it is derived from a nucleic acid precursor, it is a regulatory nucleotide. Adenlyl cyclase is the enzyme, which converts the AMP to cAMP. The cyclicAMP binds with inactive cyclic AMP receptor Protein (also known as CRP or CAP) and make it active form.
If given the choice of sugars to metabolize, E. coli will use glucose first and then other sugars. When levels of glucose are high, there is no need to express high levels of the enzymes needed for the metabolism of lactose or other sugars, which yield less energy than glucose. High levels of intracellular glucose result in low levels of cAMP. Low levels of cAMP mean very few cAMP–CAP complexes. When intracellular levels of glucose drop and other sugars must be metabolized, levels of cAMP increase. Increased cAMP levels mean there are more cAMP–CAP complexes. cAMP–CAP binds to a specific site on the DNA that is located adjacent to the promoter for the lac genes and adjacent to promoters controlling the expression of other sugar metabolizing operons affected by cAMP–CAP. Thus, the cAMP-CAP regulates lac operon positively.
Principle of Positive regulation
When glucose enters the cell, the cyclic AMP level is lowered, CRP protein cannot bind DNA, and RNA polymerase fails to bind to the promoters of operons subject to catabolite repression. Thus, catabolite repression is an indirect result of the presence of a better energy source (glucose). The direct cause of catabolite repression is a low level of cyclic AMP. The level of cyclic AMP must be high enough for the CRP protein to bind to the CRP binding site and regulates the lac operon positively.
Positive regulation could be inducible or repressive. Watch the animation (Youtube)
Nice explanation about the gene regulation
Nice explanation about the gene regulation
(Watch this youtube video for comprehensive understanding about gene regulation)
(Watch this youtube video for comprehensive understanding about gene regulation)
SUMMARY
- Many mechanisms regulate gene activity. The goal is to express genes only when needed.
- The catabolic machinery needed to metabolize lactose only needs be available when lactose is present. Likewise, tryptophan only needs to be made when the cell runs out of this amino acid.
- Transcriptional regulation is frequently observed because transcription is the first step in expressing a gene.
- Transcriptional regulation for the lac and trp operons involves a repressor, LacI or TrpR, respectively.
- LacI represses transcription when not bound by the inducer lactose. In contrast, TrpR represses transcription only when bound by tryptophan. Both of these operons have at least one additional regulatory feature.
- For the lac operon, its transcription is positively regulated by cAMP–CAP.
Report abuse