Bacteriophage_lambda_.ppt
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The document describes the life cycle choices - lysis vs lysogeny - of bacteriophage lambda that infects E. coli. During lysis, the phage DNA replicates and causes the host cell to burst, releasing progeny virus. In lysogeny, the phage DNA integrates into the host genome and remains dormant. Gene expression is regulated by transcriptional switches that determine whether lytic or lysogenic development occurs.
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Regulation in lac operon
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The lac operon is an example of nagatively controlled inducible operon.
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The trp operon of E. coli is regulated by two mechanisms - repression and attenuation. Repression involves the trp repressor binding to the operator and reducing transcription 70-fold when tryptophan is present. Attenuation determines whether transcription terminates early in the leader region or continues, providing an additional 10-fold reduction when tryptophan levels are high. Together these mechanisms provide up to 700-fold regulation to precisely control tryptophan synthesis based on cellular need.
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Regulation in lac operon
The lac operon contains three genes - lacZ, lacY, and lacA - that encode proteins involved in lactose metabolism. The activity of the lac operon promoter is regulated by the Lac repressor and CAP proteins. Negative regulation occurs when the Lac repressor binds to the operator region and prevents RNA polymerase from binding and initiating transcription. Positive regulation happens when the CAP-cAMP complex binds near the promoter and stimulates RNA polymerase binding, increasing transcription. Together, these two types of regulation allow the lac operon to be turned on or off depending on the presence of lactose in the environment.
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transposons
Transposable elements, also known as jumping genes, are segments of DNA that can change positions within a genome. There are two main types: transposons, which can carry additional genes, and retrotransposons, which move via an RNA intermediate. Transposition occurs through a "cut and paste" mechanism - the transposon is cut out of the donor DNA and inserted elsewhere, sometimes duplicating itself in the process. This movement can cause mutations by disrupting genes. Transposons have been useful for genetic engineering and mutagenesis experiments.
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Bacterial transformation involves bacteria taking up foreign DNA from their environment. This process allows bacteria to horizontally transfer genes. Transformation is used in DNA cloning to introduce plasmids containing genes of interest into bacteria. The bacteria are then selected using antibiotics to identify those containing the plasmid, which can then be used to produce large quantities of the gene or its protein product.
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The tryptophan operon regulates the biosynthesis of tryptophan in E. coli through transcriptional attenuation and repression. It contains five genes encoding the enzymes needed to synthesize tryptophan. When tryptophan levels are high, the tryptophan repressor binds to the operator site, preventing transcription. Additionally, a regulatory region can form a terminator stem-loop structure to halt transcription if tryptophan tRNA levels are high during translation of the leader mRNA sequence. However, if tryptophan levels are low, the terminator structure does not form and transcription of the operon proceeds.
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The document summarizes key aspects of bacteriophage lambda. It describes that lambda can enter either the lytic cycle or lysogenic cycle. The lytic cycle involves a cascade of gene expression controlled by regulators that ultimately leads to bacterial lysis and phage particle release. The lysogenic cycle is maintained by the lambda repressor protein, which binds as a dimer to three operator sites and prevents expression of genes needed for the lytic cycle. The repressor uses a helix-turn-helix motif for specific DNA binding and binds cooperatively to operators to maintain lysogeny through an autoregulatory circuit.
Arabinose Operon
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Lac operon
lac operon is a negatively controlled inducible operon.E.coli can use lactose as a source of carbon.
The enzymes required for the use of lactose as a source of carbon are synthesised only when the lactose is available as carbon source.
The lac operon is an example of nagatively controlled inducible operon.
Structure
The lac operon consists of 5 structural units.
Promoter
Operator
Structural genes
CAP binding sites
Regulatory gene
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Transduction is the transfer of genetic material between bacteria through bacteriophages. It was discovered in 1952 by Joshua Lederberg and Norton Zinder during their experiments with Salmonella bacteria. There are two types of transduction: generalized transduction, which occurs when a lytic phage picks up bacterial DNA during virus assembly and transfers it to another bacteria; and specialized transduction, which involves the transfer of bacterial DNA adjacent to the phage genome when a temperate phage enters the lysogenic cycle. Transduction contributes to bacterial evolution and genetic diversity by allowing for the lateral transfer of genes between distantly related species.
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Plasmids are classified as either stringent or relaxed based on their copy number in bacterial cells. Stringent plasmids exist in low copy numbers (<100 copies/cell) and rely on the bacterial genome for replication and segregation. Relaxed plasmids exist in high copy numbers (>100 copies/cell) and replicate independently of the bacterial genome. Relaxed plasmids include ColE1, which uses an antisense RNA mechanism to regulate its copy number based on plasmid concentration in the cell.
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Gene expression in bacteria and bacteriophages
1. The document discusses gene regulation in bacteria, focusing on the lac operon in E. coli. which controls lactose metabolism. 2. The lac operon contains 3 genes - lacZ, lacY, lacA - that are regulated together. When lactose is present, it is broken down into allolactose, which binds the lac repressor and prevents it from blocking transcription of the operon. 3. Experiments by Jacob and Monod identified mutations affecting regulation, including mutations in the operator or repressor gene that result in constitutive expression of the lac genes.
Regulation of lac operon positive nd negative
The document summarizes the regulation of the lac operon in E. coli. It describes how the lac operon can be regulated both positively and negatively in response to the presence of lactose or glucose. In negative regulation, the lac repressor binds to the operator site in the absence of lactose, preventing transcription. In the presence of lactose, it binds to allolactose and dissociates from DNA, allowing transcription. Positive regulation involves cAMP and the catabolite activator protein activating transcription in the absence of glucose.
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The bacteriophage lambda can undergo either the lytic or lysogenic cycle. In the lytic cycle, infection leads to production of progeny phage and lysis of the host cell. In the lysogenic cycle, the phage DNA integrates into the host genome where it remains inactive until induced to enter the lytic cycle. Key genes involved include cI which promotes lysogeny, and cro which promotes lysis. The decision depends on the relative levels of these genes - high cro leads to lysis while high cI leads to lysogeny.
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A bacteriophage is a virus that infects bacteria. Lambda phage is a temperate bacteriophage that has two life cycle choices: lytic and lysogenic. During lysogeny, the lambda repressor binds to the operator region (OR) on the phage DNA and represses transcription of lytic genes, allowing the phage genome to remain dormant as a prophage integrated into the bacterial chromosome.Artificial chromosomes - YAC and BAC
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Lytic and lysogenic cascade
1) The lambda bacteriophage can enter either the lytic cycle or lysogenic cycle in infected E. coli cells.
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The document discusses regulation of gene expression. It describes how gene expression involves transcription of DNA into mRNA which is then translated into proteins. Regulation of gene expression is important for organisms to adapt to their environment and involves mechanisms like transcription control, RNA processing, translation control and protein modification. Key examples discussed are the lac operon and lambda phage switch which demonstrate transcriptional regulation through repressor and activator proteins. The role of epigenetic factors like DNA and histone methylation and acetylation in long-term stable regulation of gene expression is also summarized.
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promoter region
The promoter region is a DNA sequence located near the transcription start site of a gene that initiates transcription by RNA polymerase binding to it. Promoters contain response elements that provide binding sites for RNA polymerase and transcription factors. There are three main portions of a promoter: the core promoter contains the RNA polymerase binding site and transcription start site; the proximal promoter contains regulatory elements; and the distal promoter further upstream also contains regulatory elements. In prokaryotes, the core promoter contains -10 and -35 sequences recognized by sigma factors associated with RNA polymerase. In eukaryotes, transcription requires many general transcription factors and RNA polymerase is specific to the type of RNA transcribed. Regulation of transcription involves repressors and activators that bind
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The document discusses various mechanisms of gene expression and regulation in prokaryotes and eukaryotes. It explains that gene expression involves the transcription of DNA into mRNA and the translation of mRNA into protein. In prokaryotes, genes may be organized into operons and regulated through repression and induction. The lactose and tryptophan operons are discussed as examples. Eukaryotes have additional complexities in gene expression including RNA processing, alternative splicing, and transport of mRNA. Gene expression is regulated at transcriptional, post-transcriptional, translational, and post-translational levels.
Translation
This presentation explains the fundamentals of Genetic Code, Protein synthesis mechanism and Antibiotics that inhibits at various stages of Translation.
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Bacteriophage_lambda_.ppt
- 1. Transcriptional switches can regulate cellular decisions
- 2. Lysis or Lysogeny • Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium • Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively – No progeny – No lysis of the host – Can subsequently lyse (lysogeny) • Bacteriophage lambda can do either.
- 4. The phage genome integrated into the host bacterial genome is a prophage. Bacterium carrying the prophage is a lysogen. Lysogens are immune to further infection by similar phage because the phage functions are repressed in trans. Induction of the lysogen leads to excision of the prophage, replication of the phage DNA, and lysis of the host bacterium.
- 5. Genes are clustered by function in the lambda genome Recombination Control region Replication Lysis Virus head &tail origin oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J promoter operator terminator Late control cos Not to scale!
- 6. Immediate early transcription Transcription by E. coli RNA polymerase initiates at strong promoters PR , PR’, and PL , and terminates at t’s. 6S RNA oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J N Cro
- 7. Antitermination by N protein leads to early gene expression Pint PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J N N N N protein Cro 6S RNA CIII Recombination proteins CII Replication proteins Q protein
- 8. Lytic cascade: Cro turns off cI, Q protein action leads to late gene expression oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J Cro Cro Q Lytic functions Replication proteins Viral head & tail proteins
- 9. Late stage of lytic cascade High concentrations of Cro turn off PR and PL . Abundant expression from PR’. oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J Cro Cro Q Lytic functions Viral head & tail proteins
- 10. + Lysogeny: CII and CIII stimulate expression of cI to make repressor oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J CIII CII CI + Repressor PRE = promoter for repression establishment Int tint CII
- 11. Lysogeny: Repressor turns off transcription oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J CI Repressor PRM = promoter for repression maintenance CI CI Activated by Repressor binding to oR1 & oR2
- 12. Regulatory mutants of lambda Clear plaque mutants Virulent mutants (vir) Need wild type for lysogeny: Establishment Maintenance cI Yes Yes cII Yes No cIII Yes No Act in trans Act in cis : are double mutants in oR &/or oL
- 13. oR1 oR2 oR : TTGACT GATAAT -10 -35 ATAGAT 5’ TTAGAT 5’ -10 -35 oR3 cro N PR PRM
- 14. operator N-terminus: DNA binding; Helix-Turn-Helix motif C-terminal domain: protein-protein interaction; dimerization and cooperativity Connector operator operator oR1 oR2 l repressor is a dimer; monomer has 236 amino acids. l repressor can bind cooperatively to operator sub-sites.
- 15. Place a convenient reporter gene under control of the regulatory elements being studied Use a known regulatory region to control the trans-acting regulatory element
- 16. lac p, o l cI l pR , OR lacZ Place l cI gene under lac control. Use lacZ as a reporter. E. coli with lac repressor, no lacZ. Control amount of l repressor by [IPTG]. See effect of l repressor by b-galactosidase activity 321
- 17. lac p, o l cI l pR , OR lacZ b-galactosidase [IPTG] l repressor l repressor acts cooperatively.
- 18. lac p, o l cI l pRM , OR lacZ b-galactosidase [IPTG] l repressor 123 l repressor at oR1 and oR2 stimulates transcription from pRM.
- 19. oR1 oR2 -10 -35 -10 -35 oR3 cro N PR PRM 2 dimers of Repressor, bound cooperatively RNA Pol = operator -10 -35 = promoter
- 20. lysis or lysogeny (cI or Cro?) ? Both lysis and lysogeny: PR, PL, PR’ active : synthesize N, Cro antitermination by N : synthesize cIII, cII, Q Lysis: Low [Cro] : binds OR3, shuts off PRM (cI) High [Cro] : shuts off PR and PL antitermination by Q + activation of PR’ by Cro
- 21. lysis or lysogeny (cI or Cro?) ? Lysis and lysogeny : PR, PL, PR’ active : synthesize N, Cro antitermination by N : synthesize cIII, cII, Q Lysogeny: cII stimulate expression from PRE (cI repressor) and PINT (integrase) cIII stabilizes cII cI repressor shuts off PR, PL, PR’ (no lytic functions), stimulates PRM
- 22. Temperate and lytic phage have a different plaque morphology Temperate phage generate turbid plaques lysogenized cells lysed cells uninfected cells lysed cells Mutants of phage that have lost the capacity to lysogenize form clear plaques Lytic phage: clear plaques
- 23. Induction and immunity of lysogens l A l lysogen l Spontaneously, 1/1000 lysogens will induce, i.e. the l prophage will excise, replicate and lyse the cell. UV treatment leads to induction of virtually all lysogens in a culture. Lysogens are immune to further infection with similar (lambdoid) phage + l