Virus and Prokaryotic Gene Regulation - 1

Transcription

1 Virus and Prokaryotic Gene Regulation - 1 We have discussed the molecular structure of DNA and its function in DNA duplication and in transcription and protein synthesis. We now turn to how cells regulate gene expression. Gene regulation is one of the most active areas of genetic research. Some of the answers to how genes are regulated are coming from work on recombinant DNA research, some from genetics, including the effect of mutations on gene expression, and some from research on disease. Much is coming from our increasing knowledge of cancers and the failure of the body to control cell division in cancer formation. It is important for cells to be able to control gene activity. We have genetic information for thousands of proteins. We do not want to synthesize enzymes that are not needed, nor do we want to synthesize molecules in greater quantity than needed. Our discussion on gene regulation will include looking how gene expression is regulated in viruses, in prokaryotes and in eukaryotes from regulating access to DNA to affecting the post translation processing of proteins. Viruses are mobile genetic elements or infectious genetic elements that regulate gene expression by using the DNA replication, transcription and translation processes of their host cells to synthesis new virus genetic material and coatings. Prokaryotes, in general, control genes for rapid response to their environment. By selectively activating (inducing) or inhibiting gene activity, bacterial cells can take advantage of changing conditions. For eukaryotes, gene regulation is tied to maintaining homeostasis a consistent internal environment in the face of ever-changing external conditions. Multicellular organisms require different genes at different times of growth and development in different tissues. We have more complex controls of gene expression to ensure that genes function selectively and appropriately in our different tissues from the zygote through all stages of growth, development and maturation. Gene control is exerted chemically in two general ways: affecting molecules that interact with DNA, RNA and/or the polypeptide chains, or controlling the synthesis of an enzyme or the activity of an enzyme in the cell.

2 Virus and Prokaryotic Gene Regulation - 2 Gene controls can be positive inducing or activating gene activity, or negative repressing gene activity. Recall that initiation of transcription involves a set of transcription factors that locate the promoter region of the gene to be transcribed and position RNA polymerase on the DNA for transcription. Once fixed into position, RNA polymerase catalyzes transcription. In negative control, the gene is normally transcribed, with the promoter region readily accessible to transcription factors. To stop transcription, a repressor molecule must bind to the DNA at a repressor site blocking transcription. In positive gene regulation, an activator must bind to an activator site in order for transcription to occur. Negative Gene Regulation Positive Gene Regulation We will now turn to examples of gene regulation in viruses, prokaryotes and eukaryotes.

3 Virus and Prokaryotic Gene Regulation - 3 Viruses and Virus Genetics Viruses are particles composed of a genetic molecule surrounded by a protein coating called a capsid and sometimes a membranous "envelope" of host cell membrane. By convention, a virus outside of its host may be called a virion and viruses that invade bacteria are called bacteriophages or phages. T4 Bacteriophage on E. coli Viruses Emerging from Human Cell A virus is not a cell and does not carry out most processes characteristic of life. Viruses do not have membranes to regulate movement of materials into and out of themselves and have no nutrient metabolism. Viruses are also not self-replicating (a requirement for life), but are capable of turning their host genetic molecules into virus making "machines". As such, they are intracellular "parasitic" chemicals as well as mobile genetic elements. In this section, we will look at the viral reproductive cycle and the how viruses regulate their gene expression. Viruses are categorized by their kind of genetic molecule, their surface coatings, and by the kinds of organisms they infect. The Viral Genome Virus genetic material is more variable than that of living organisms. The virus genetic molecule has from 4 to several hundred genes and is either a single linear nucleic acid molecule or a circular nucleic acid molecule. The genetic molecule can be double or single-stranded DNA or RNA. Some examples of virus diversity are shown below.

4 Virus and Prokaryotic Gene Regulation - 4 Virus Reproduction and Gene Regulation Since viruses cannot reproduce by themselves, they are obligate intracellular parasites. An isolated virus can do nothing until and unless it comes into contact with the appropriate host cell. Most viruses have a narrow range of hosts, generally just one or a few types of cells (or tissues) within a specific host species. A virus can identify appropriate host cells just like a substrate fits an enzyme. The virus fits into its host cell's membrane receptor. Bacteriophages have proteins in their capsid that bind to receptor molecules in their host's cell wall. Virus Lytic Cycle The "common" virus cycle is known as a lytic cycle. Once inside of the host cell, the virus reprograms the host cell into a virus-making cell. A lytic bacteriophage has a promoter sequence in its genome that attracts the host's RNA polymerase. Initially, the viral genes transcribed stop host transcription and promote activity to duplicate the viral genome, using nucleotides from degraded DNA molecules of its host cell. Later, the host's RNA polymerase transcribes the genes that promote synthesis of viral proteins of the capsid and enzymes that will lyse the host cell. This whole process can take less than 30 minutes.

5 Virus and Prokaryotic Gene Regulation - 5 Virus Lysogenic Cycle Some viruses have a lysogenic cycle rather than a lytic cycle. Lysogenic viruses enter a host cell and join their viral DNA with the host cell's DNA for an unlimited period of time. A bacteriophage in this phase is called a prophage. (A virus doing this in a eukaryotic cell is called a provirus.) A lysogenic virus can use the host cell's replication for the viral genome without destroying the host. A lysogenic phage's host bacterium will typically transcribe one gene of the viral DNA that represses the remainder of the prophage's genes from activity. In some cases, the host will transcribe additional viral DNA, with resulting biochemical and phenotypic changes to the host cell. Three of our more deadly diseases are caused by toxins coded by prophages within the infectious bacterium: diphtheria, botulism and scarlet fever. The host cell will also duplicate the viral DNA (because it's now part of the host's chromosome) when the host cell divides passing the viral instructions along to new cells. Temperate Viruses A temperate virus has both lytic and lysogenic phases. The E. coli lambda ( ) phage is a temperate phage. Temperate viruses can convert the lysogenic stage into a virulent lytic stage when an appropriate signal is provided. The signal causes the viral DNA to exit the host cell's DNA and activate the lytic cycle. In some instances, a temperate phage may, when excising from its host's DNA, carry a portion of the host's DNA with it. When new viruses from the lytic cycle stage exit the bacterium, they carry that portion of host DNA with them. When entering new bacteria in the lysogenic cycle stage, transduction, discussed earlier as one way of bacterial recombination, can occur.

6 Virus and Prokaryotic Gene Regulation - 6 Virus Transcription Control of Lytic and Lysogenic Cycles The genetic controls of the Lamba ( ) temperate phage have been studied as an example of virus gene regulation. Not surprisingly, phage is lysogenic when the host is in a favorable environment where it can grow and divide rapidly. Such environment will produce many new bacterium generations, each carrying the phage. phage becomes lytic when the host becomes unhealthy. Two virus proteins, Cro and cl monitor the "health" of their host. When the host is healthy, cl proteins accumulate to activate lysogenic gene function and repress lytic gene function. When the host is less healthy, Cro proteins accumulate, which repress lysogenic gene activity and promote lytic gene activity. Each protein acts on the promoter region of its target genes to either repress or activate the gene. The ratio of Cro to cl determines whether phage will be lysogenic or lytic. Other viruses have similar gene controls. Although much of what has been mentioned about bacterial viruses applies to both all viruses, we will want to discuss some of the things about viruses that infect other kinds of organisms as well, in particular animal viruses and plant viruses. Eukaryotic Viruses Recall that the genetic molecule of viruses may be DNA or RNA. DNA viruses may be either single-stranded or double-stranded. DNA viruses directly use the host's transcription enzymes.. RNA viruses include viruses whose RNA is translated directly or viruses whose RNA serves as a template for mrna transcription. Many plant viruses are RNA viruses as are the flu and poliovirus of humans. Retroviruses use reverse transcriptase to first produce a complementary DNA strand (and its complement) for a double-stranded DNA molecule that incorporates into the host genome as a provirus..

7 Virus and Prokaryotic Gene Regulation - 7 Eukaryotic viruses can be "naked" or have a membrane "envelope". A naked animal virion may be incorporated into its host via endocytosis, forming a membrane-coated vesicle within the host cell. As with other endocytotic vesicles, the membrane is degraded, the virion's capsid is degraded and the viral nucleic acid directs the host cell to manufacture new viral components. Newly assembled viruses will lyse the cell to exit. Enveloped viruses have glycoproteins in a viral membrane to facilitate the entry of the virus into the host cell. The viral membrane binds with the host cell membrane. Within the cytosol, the capsid and genetic molecule are dissembled and the viral genetic material directs the host cell to manufacture new viral components. The host ER is used to make new viral glycoproteins that are transported to the plasma membrane in Golgi vesicles. These vesicles merge with the plasma membrane to facilitate the exit of new viruses, now complete with a bit of the host plasma membrane and virus glycoprotein markers as its new viral envelope. Such viruses may exit without damage to the host cell. Some viruses, such as the Herpes virus, duplicate within the nucleus. Some viral DNA can remain in the host nucleus to reactivate later to infect new cells in the body Endocytotic Enveloped Influenza Virus HIV Enveloped Retrovirus Membrane Fusion

8 Virus and Prokaryotic Gene Regulation - 8 HIV A Human Retrovirus HIV is a retrovirus. Infection with HIV to date has been generally fatal, and the infection rate is, worldwide, increasing. HIV is an enveloped virus that contains two identical RNA copies and two reverse transcriptase copies, along with enzymes needed to integrate into the host cell's genome. When HIV enters a host cell, it uses its reverse transcriptase to synthesize viral DNA that can be transcribed by its host transcription complex. The viral DNA then integrates into the host cell DNA as a provirus, which becomes a permanent part of the host cell DNA. The provirus DNA can be transcribed and translated to form new HIV including RNA, reverse transcriptase, capsid proteins and glycoproteins for its envelope, and new virions are released from the host cell to infect new cells. HIV specifically targets the Helper T cells of the human immune system. Its origin has been traced to a less virulent primate virus in Africa, although multiple origin strains have been identified. HIV is a virus that rapidly mutates. HIV is transmitted through sexual fluids, infected blood or by open wound contact with blood of someone infected. Helper T-cells are critical to the activation of the specific immune responses. A macrophage that finds a foreign substance (antigen) will "sample" a bit of the antigen and become a specific antigen-marked macrophage. As antigen-marked macrophages circulate, they come into contact with Helper T-cells. This contact stimulates helper T-cells to activate a series of specific immune system responses. Helper T-cells bind to antigen-possessing B-cells to promote rapid division of specific B-cells with the antigen marker. Helper T-cell secrete interleukin-2 to promote rapid division of Cytotoxic (Killer) T-cells When Helper-T cells are destroyed by HIV, the immune system is compromised, and eventually, most infected with HIV get AIDS (acquired immune deficiency syndrome). Almost everyone with AIDS dies from a combination of opportunistic wasting diseases. The only way to minimize the risk of HIV infection is to always practice protected sex and avoid possible exposure to infected blood. A number of HIV treatments have been developed, and many infected individuals are surviving with reasonable health for several years where treatments are available. In other world areas, AIDS is spreading rapidly.

9 Virus and Prokaryotic Gene Regulation - 9 HIV Regulation and Treatments Almost every step of the HIV cycle is a target for treatment of, and blocking infection by HIV. Blocking transcription One way cells have of suppressing viral infection is to bind to viral mrna when the host cell's transcription is activated, causing RNA polymerase to detach from the virus DNA segments. This doesn't work with some viruses, including HIV. HIV has a protein, called TAT (transactivator of transcription) which binds to termination proteins blocking their ability to terminate transcription of the HIV mrna. Inhibiting TAT synthesis is just one of the HIV treatment targets. HIV Treatments Much effort has been made to treat those infected with HIV. Some of the research to date includes: The drug AZT which binds to reverse transcriptase blocking replication Use of protease inhibitors and other drugs that block production of viral proteins needed for HIV transcription, translation of HIV RNA, enzymes, capsid and virion assembly Vaccine that would insert a defective gene into the provirus Drug therapy that might block production of a critical viral protein Chemical inhibitors to block the host cell HIV entry receptors Mutations of the host cell receptors so that HIV cannot recognize them

10 Virus and Prokaryotic Gene Regulation - 10 Prokaryotic Gene Regulation Switching Genes On and Off Prokaryotes respond rapidly to whatever is in their environment relative to their needs. Recall that the typical gene codes for a polypeptide that is used to help the cell function in some way or is a structural protein. When a particular fuel molecule is in the environment, prokaryotes will synthesize the enzymes needed to process that fuel molecule. When a particular amino acid is plentiful in the environment, genes that normally code for the enzymes that catalyze the synthesis of that amino acid are blocked, so that the bacterium does not overproduce a substance not needed. The Prokaryote Operon A gene that codes for a protein product, such as an enzyme, is a structural gene (also known as a transcriptional unit). A functional gene includes the structural gene or a related set of structural genes and additional DNA segments known as the promoter and operator. This coordinated complex in prokaryotes is known as the operon and was described in 1961 by Francois Jacob and Jacques Monod who determined how lactose was metabolized in E coli. An operon has three parts: promoter, operator and structural gene(s). In addition there are associated regulatory genes that control whether the gene is transcribed or not. The Operon Complex 1. Promoter The promoter is recognized by RNA polymerase as the place to start transcription. 2. Operator The operator controls RNA polymerase's access to the promoter, and is usually located within the promoter or between the promoter and the transcribable gene. The operator essentially switches on or off the gene. 3. Structural (Transcribable) Gene The structural gene (or set of genes) codes for the needed polypeptide(s).

11 Virus and Prokaryotic Gene Regulation - 11 Regulatory Genes Coding for the On-Off Switch Recall that during our introduction to gene regulation, we described both positive and negative controls of gene activity. The regulatory gene, located at some distance from the operon on the DNA, usually codes for a gene repressor that functions as the on-off switch for the operator site of the operon complex. To complicate things, a repressor generally works with controller molecules, which are generally some cellular substance that control the repressor. A controller can activate a repressor, which then binds to the operator region of the gene, stopping, or repressing, gene activity. A controller that activates a repressor is sometimes called a co-repressor. A repressor may normally be sitting on the operator region of the gene. A controller can remove or deactivate the repressor, which activates, or induces, gene activity. Both options exist, because some genes are naturally "on" and some are naturally "off". Hence, we have both inducible operons, those that must be "induced" to transcribe, and repressible operons, those that must be "repressed" to stop transcription. (This sounds weird, but we have vocabulary that addresses these options.) Inducible and Repressible Operons An inducible operon is one whose structural gene is normally off, blocked by its repressor. Its controller substance attaches to the repressor molecule, removing it from blocking the gene (hence stopping its repression). The gene can then be "on", hence transcribed, until the repressor once again attaches to the gene's operator. A repressible operon is one whose gene is normally "on". When the controller substance attaches to the repressor molecule (that has not been attached to the operator), the repressor binds to the operator, blocking the structural gene and turning it off. In both inducible and repressible operons, the gene is off when the repressor is attached to its controller. The difference is where the repressor is located.

12 Virus and Prokaryotic Gene Regulation - 12 Inducible Operon Repressor active on gene Gene is off. Controller removes repressor from gene to activate the gene. Gene is then turned on so long as the controller substance remains attached to the repressor molecule. Repressible Operon Gene is normally on, and the repressor is not attached to it. Controller binds to repressor, which then attaches to the operator blocking the structural gene. Gene is turned off. The Lactose Operon An Inducible Operon Jacob and Monod studied the lactose operon that controls lactase synthesis in E. coli. In the lactose operon, the substrate, allolactose (an isomer of lactose), attaches to a repressor protein that normally sits on the operator region of the gene. In the absence of lactose (the controller), the repressor sitting on the operator inhibits transcription by blocking RNA polymerase from attaching to the promoter. Lactose absent, repressor active, operon off When the substrate, allolactose (the controller), attaches to the repressor molecule that sits on the operator region of the gene, the repressor is removed from the gene. RNA polymerase can attach to the promoter and the genes that code for the three enzymes to digest lactose are transcribed.

13 Virus and Prokaryotic Gene Regulation - 13 Lactose present, repressor inactive, operon on When the enzymes are synthesized, the lactose is degraded, including the allolactose molecules attached to the repressor. When allolactose is no longer available to bind to the repressor protein, the repressor shuts down the promoter (by sitting on the operator), which stops transcription. This, again, is a negative control mechanism, because the promoter is blocked from activating the operator by the repressor. The lactose operon is an example of gene regulation of inducible enzymes, because the presence of the substrate of the metabolic pathway can induce the synthesis of the enzyme. Because allolactose induces transcription, the lactose operon is called an inducible operon (or in some sense, a derepressable operon because lactose stops the repressor from its activity). One view of the Lactose Operon

14 Virus and Prokaryotic Gene Regulation - 14 The Tryptophan Operon A Repressible Operon All cells need the amino acid, tryptophan. Bacteria, such as E. coli, have the enzymes needed to synthesize tryptophan, and the genes that code for these enzymes are normally active. However, if tryptophan is in E. coli's environment, transcription is halted, and the enzymes needed to manufacture tryptophan in the bacterial cell are not synthesized. A cellular product, in this case, tryptophan, can function to inhibit transcription, and is an example of the feedback inhibition common in cell homeostasis. High concentration of tryptophan stops the transcription of the set of enzymes that lead to tryptophan synthesis. Tryptophan does so by binding to an allosteric (non-active) site on the tryptophan repressor. This alters the shape of the repressor protein so that the operator blocks the attachment of RNA polymerase to the promoter. The tryptophan operon is an example of gene regulation of repressible enzymes, because the presence of the product of the metabolic pathway represses (or stops) the synthesis of the enzyme(s) needed to synthesize it. Technically, the controller, tryptophan, is called a co-repressor because it works with the repressor protein to block transcription, and the tryptophan operon is a repressible operon. This is a negative control mechanism because the repressor blocks transcription. Gene Transcription when Tryptophan Absent Transcription Repressed when Tryptophan Present

15 Virus and Prokaryotic Gene Regulation - 15 Both the lactose operon and the tryptophan operon block gene activity. The presence of tryptophan actively blocks the operator of the gene that codes for the production of tryptophan. Lactose substrate removes the repressor from blocking transcription of the genes that code for lactose-degrading enzymes. Metabolically, inducible enzymes, such as those synthesized with the lactose operon, are often found in catabolic pathways. It makes biological sense to have the target molecule (or substrate) function as the controller for the activation of the genes. When the substrate is gone, you no longer need to synthesize the enzymes to degrade it. Conversely, repressible enzymes, such as those needed for the synthesis of tryptophan, are often in anabolic pathways. In this case, the product serves as the controller molecule to block enzyme synthesis. When you have the product, you no longer need to synthesize the enzymes. Gene Enhancement Catabolite Repression and Activator Proteins Gene activity can also be regulated by molecules that enhance a promoter's receptivity to RNA polymerase. Such molecules are called activators. Without the activator molecule present, the gene is poorly transcribed, if at all. Catabolic pathways often have gene activators that promote synthesis of enzymes utilizing alternative fuel molecules in the absence of a preferred fuel source, but repress gene activity when preferred fuel molecules are available. This is known as catabolite repression. The lactose operon is one example of catabolite repression. (It may seem strange to use a term, catabolite repression, for an enhancement of a promoter, but this is biology.) The lactose inducible operon works in tandem with an activator, CRP (cyclic AMP receptor {or response} protein), also known as CAP (catabolite activator protein) that monitors the amount of glucose in the cell. Since glucose is the preferred fuel molecule in cell respiration, high levels of glucose in the cell block the lactose operon no matter how much lactose is present. There is no need to degrade lactose for fuel if there is plenty of glucose fuel available. This is also known as glucose repression, because the presence of glucose represses gene activity for other fuels.

16 Virus and Prokaryotic Gene Regulation - 16 However, when glucose levels are low in the cell, camp (cyclic adenosine monophosphate), an important secondary messenger in cell communications, accumulates. camp binds to the allosteric site of CAP forming a CAP-cAMP (CRPcAMP) complex. CAP-cAMP binds to a site next to the lactose operon promoter and makes it easier for RNA polymerase to bind to the promoter region enhancing transcription of the lactase enzymes when lactose is present in the cell's environment to remove the lactose repressor molecule. Activation of the lactose operon relative to available fuels Although we have used the example of CAP (CRP) enhancement of lactase gene activity, CAP (CRP) is a regulator for many catabolic pathways in E. coli, working with as many as 100 genes.

17 Virus and Prokaryotic Gene Regulation - 17 Some Human Pathogenic Viruses