Control of gene expression in phages: Lytic cycle & Lysogenic cycle
Introduction: Control of gene expression in phages
Phages, also known as bacteriophages are viruses that infect bacteria like E.coli. Bacteriophages either have DNA or RNA as their genetic material. They are linear or circular in the configuration as a single or double-stranded form. λ-phage has a dsDNA genome which is linear with a size of about 48.5kb in the capsid and circular when it is in the host. After infection, the linear genome is converted into a circular form by binding to two cos sites. Control of gene expression in phages is mainly driven by two kinds of cycles.
A phage usually follows one of the two life cycles called as lytic cycle and lysogenic cycle:
1)Lytic cycle –
It is a cycle of viral reproduction which destroys the infected cell. When only a lytic cycle is used by the bacteriophage it is called a virulent phage.
- Attachment – The first stage in the lytic cycle is an attachment which means the phage is attached surface of the host cell to inject its DNA into the bacterial cell.
- Penetration – Becteriophage injects its DNA into the host cell by penetration through the cell membrane.
- Transcription – Penetration is followed by the transcription in which the host cell DNA is degraded and the cell’s metabolism is directed to initiate the phage biosynthesis. The phage uses the overall machinery of the host cell for the synthesis of different viral particles.
- Replication – In this process, new phage DNA and proteins are synthesized.
- Maturation – The replicated viral material is going to assemble to form a full viral phage. After maturation of phage, the lysis of the host cell takes place and the new phage that has been made inside the bacterial cell will be released into the environment.
2)Lysogenic cycle –
When a phage infects the surface of bacteria through attachment it penetrates its DNA into the bacterial cell. After penetrating its DNA into the bacterial cell, lysogeny is characterized by the integration of bacteriophage DNA into the host bacterium genome. In this condition, the bacterium is not destroyed and continues living and reproducing normally. When the genome of a bacteriophage is integrated into the host genome it is called a prophage. Prophage can be transmitted to the daughter cell at each subsequent cell division. This integrated prophage replicates when bacterial DNA replicates.
How gene expression is regulated in phages?
In order to understand the Control of gene expression in phages, we need to know the temperate phage. The phage in which both lytic and lysogenic cycles are present is called temperate phage. The regulation of gene expression in phages is all about how the lytic cycle gets switched to the lysogenic cycle and vice-versa. λ-phage is the best example of a temperate phage. It can switch between the lytic cycle and the lysogenic cycle.
There are three classes of genes in the phage genome that regulate whether the lytic or lysogenic cycle will emerge.
- Immediate early genes – 1)Gene “N” -Antiterminator or activator of transcription. 2) Gene “cro” – Forms cro repressor protein.
- Delayed early genes – 1)cII – Activator of Transcription of cI 2)cIII – a structural mimic of cII and stabilizes cII 3)int (integrate) – specific for integration in the host genome 4)xis – excision of prophage from the host genome by enzyme Excisionase. 5)O & P – Replication of DNA 6) Q – for the expression of late genes.
- Late genes – 1) A to Z – Head and tail synthesis. 2)S and R – genes for lysis.
Promoters and operators: Phage lambda has two early transcription units i.e. Leftward and rightward.
- PL – Left promoter (initiate transcription of N)
- PR – Right promoter ( initiate transcription of cro)
- PRE – Promoter for repressor establishment (helps cI repressor for establishment )
- PI – Promoter of integration
- PRM – Promoter of λ-repressor maintenance
- OR1, OR2, OR3 – operator regions of PR & PRM
- OL1, OL2, OL3 – operator regions of PL
Let’s see how these genes help in the regulation of gene expression in the lytic and lysogenic cycle:
When bacteriophage infects the host cell the promoter PL and PR start the transcription. Promoter PL and PR are responsible for the transcription of genes N and cro respectively. The main function of cro is to induce the lytic cycle by binding to the RNA polymerase and repressing the cI repressor and N acts as Antiterminator which binds to RNA polymerase and prevents transcriptional termination. Since the transcription of genes N and cro started early they are called immediate early genes.
After the transcription of N and cro, due to the action of the Antiterminator(N), cII and cIII genes also get transcribed by PR and PL respectively. These genes are called delayed early genes. Gene cII and cIII help in the lysogenic cycle.
How is the lysogenic cycle established?
Promoter PRE establishes the cI repressor with the help of cII and cIII. cII is the activator of transcription of cI but it is more unstable so it will be easily degraded by cellular proteases made by the host bacterium. To avoid this, cIII binds to cII and degrade the cellular protease of the host bacterium and prevent the degradation of cII. Along with RNA polymerase, this cII-cIII complex synthesizes the cI repressor protein.
cI repressor protein is a dimer made up of 236 amino acids. It has C and N terminals. C terminal is responsible for dimerization and N terminal is for DNA-binding. As cI is a repressor protein its main function is to repress cro and induce a lysogenic cycle.
How cI repressor inhibits the genes required for the lytic cycle? The λ-repressor or cI repressor binds to operators that are adjacent to PR and PL. Both PR and PL have three operator regions i.e. OR1, OR2, OR3, and OL1OL1, OL2, OL3 respectively. λ-repressor has the highest affinity to OR1 then OR2 and OR3. (OR2 & OR3 is an operator for PRM also as it is adjacent to PR). OR1>OR2>OR3, OL1>OL2>OL3
λ-repressor binds to the OR1 region and inhibits PR to initiate transcription so there will be no production of cro protein and by repressing the expression of cro protein lytic cycle will be inhibited and the lysogenic cycle will be established.
When cI binds to the OR2 region it will stimulate the expression of cI with the help of PRM. (PRM & PR present adjacent to each other. If PR is blocked PRM initiates the expression. But they both can’t express simultaneously). When cI binds to the OR3 region it represses its synthesis. This is why the cI repressor acts as an activator as well as a repressor because it activates its synthesis and represses it also when its amount becomes high.
For the maintenance of the lysogenic state, λ- repressor blocks PR and PL but activates PRM which initiates transcription by RNA polymerase and synthesis of cI mRNA continued.
In the lysogenic cycle, there is a synthesis of the prophage. Prophage is made by the integration of the viral genome into the bacterial genome by the enzyme integrase. This integrase protein is synthesized by the int gene present in the viral genome. The cII along with RNA polymerase bind to the promoter site of the int gene called PI (promoter of integration) and integrase protein is synthesized.
Why int gene is transcribed by PI promoter and not by PL promoter of N antitermination? This is because even though the N Antitermination complex transcribed the int gene, it was demonstrated that sib inhibits the expression of the int gene from PL but not from PI. sib is a cis-acting, retro regular downstream of the int gene.
The sib site forms the large stem-loop structure in the RNA which is sensitive to cleavage by RNaseIII. So the cleavage of the transcript initiated by PL promoter at sib by RNaseIII prevents int protein synthesis. Therefore sib is acting as a retroregulator by inhibiting PL to transcribe the int gene.
How is the lytic cycle established?
In the lytic cycle, two regulators cro and N transcribe by binding to RNA polymerase with the help of promoters PR and PL and form cro mRNA and N mRNA.
During the lytic cycle, the cro protein controls the switch. cro protein is a dimer & it has the highest affinity to OR3 then the same for OR2 and OR1. (OR3>OR2=OR1, OL3>OL2=OL1)
When cro protein bind to OR3, it will block transcription from PRM by repressing the expression of the cI repressor so the lysogenic cycle will be switched off. Late genes are expressed in the lytic cycle. Q gene is responsible for capsid formation and the lysis of the bacterial cell. It acts as an antiterminator that permits transcription of late genes through another promoter, PR’. PR’ controls a very large operon that encodes the protein necessary for the assembly of phage coat, packaging of DNA, and lysis of bacterial cells.
To summarize all this, in the lysogenic cycle, the cI repressor blocks the transcription from PR and PL by binding to their operators and blocking expression of cro which allows RNA polymerase to bind to PRM and transcribe to form cI mRNA. While in the lytic cycle, cro represses the expression of the cI repressor by binding to the operator of PRM i.e.OR3 and inhibiting RNA polymerase to bind to PRM. So, RNA polymerase attaches to PR and PL and transcribes cro gene and N gene to form cro mRNA and N mRNA respectively.
cI repressor ➡ repress cro ➡ Lysogenic cycle
cro repressor ➡ repress cI ➡ Lytic cycle
Factors that influence the choice between two cycles :
cII protein plays a key role in directing cI repressor to the lysogenic or lytic cycle. The cellular protein HFLA produced by bacteria forms FtsH protease that degrades the cII protein. These proteases are produced depending on environmental conditions. If the growth conditions are very favourable, the level of HFLA is high and it will degrade the cII protein and the cI repressor will not be synthesized so the lysogenic cycle will be switched off. Instead high levels of cro protein allow the lytic cycle to proceed. Thus, favourable growth conditions promote the lytic cycle because a sufficient supply of nutrients is necessary for the synthesis of new bacteriophages.
If the growth conditions are unfavourable that means if nutrients are limiting, the level of HFLA is low. Due to this cII protein builds up more quickly than cro and turns on PRE, forming the cI repressor. Thus, unfavourable growth conditions promote the lysogenic cycle because no sufficient nutrients are present for the production of new bacteriophages.
High nutrients(HFLA)➡ cII degraded➡ high cro level➡ Lytic cycle
Low nutrients (HFLA)➡ cII builds up quickly ➡activate PRE ➡cI made ➡ Lysogenic cycle
Certain conditions like exposure to UV light can also favour the induction of the lytic cycle. A cellular protein recA(a protein involved in DNA recombination) activates to become a protease by detecting the DNA damage. This protease cleaves cI repressor and inactivates it. Therefore, cro protein will accumulate and favours the lytic cycle.
How is gene expression controlled?
Gene expression means the transcription of a gene into mRNA and it’s a translation into protein i.e. when a gene leads to the formation of protein and plays function it is said to be an expression of the gene. Various factors control how much a gene is transcribed, they are called transcription factors. These are the regulatory proteins that regulate transcription. The expression of genes is mostly controlled at the level of transcription but in eukaryotes, it is controlled at both transcriptional and post-transcriptional levels. The binding of regulatory proteins to the specific sites of DNA(promoters) controls the gene expression. Transcription factors like activators and repressors help specific genes to turn “on” and “off”. Activators activate the transcription while repressors inhibit the transcription.
What factors control gene expression?
The action of various factors responsible for the control of gene expressions like transcription factors, non-coding RNAs, chromatin remodeling, modifications in the structure of histones, and DNA(methylation, phosphorylation, acetylation ). Transcription factors like activators and repressors help genes to run “on” or off” while non-coding RNAs help in gene silencing by degradation of mRNA. Chromatin remodelling, DNA, and histone modifications inhibit or activate the expression of genes by altering the chromatin structure.
How is gene expression controlled in bacteria?
Bacterial genes found in clusters(operon) means they are expressed in groups with the help of a single promoter. Each operon contains specific regulatory proteins that bind to regulatory DNA sequences and promote or inhibit transcription. Some of the operons in bacteria are inducible or some are repressible. The presence of a small molecule can turn on an inducible operon. The regulatory proteins like activators bound to their DNA binding sites and increases transcription by helping RNA polymerase to attach to the operator. When the repressor bind to the operator it reduces the transcription by blocking RNA polymerase from moving forward to DNA.
What does acetylation of histones do?
Histone modification is an epigenetic modification that regulates the expression of genes by closing or opening the structure of chromatin. In histone acetylation, the acetyl group is added usually to lysine residues at the N-terminus by histone acetyltransferase(HAT). HAT proteins attach the acetyl group to histone proteins which neutralizes their positive charge. DNA is less attracted to histones when histones lose their positive charge and the bond between DNA and histones is weakened. DNA is decondensed due to weak bonds and allows transcription factors to bind and initiate transcription. Therefore, histone acetylation leads to the activation of transcription.
What are two ways of controlling gene expression in bacteria?
The control of expression of genes in bacteria is carried by two types of operon i.e. Inducible operon and repressible operon. Operon is a cluster of functional genes regulated under a common promoter. Inducible operon kept turned off in normal conditions and only activate when the repressor bind to the inducer. The transcription is turned on in an inducible operon. Inducible operon only turns on in the presence of their substrate. While repressible operon is always turned on by default and repressor is inactive under normal conditions. The binding of the co-repressor to the repressor turns off the operon and transcription turns off.
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