Chapter 20 Genome Defense Figure 2001 Antisense RNA Can Base Pair With mRNA mRNA is normally made using the noncoding strand of DNA as a template Such mRNA is also known as sense RNA ID: 999023
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1. Unit 4: Regulating Gene ExpressionChapter 20 - Genome Defense
2. Figure 20.01. Antisense RNA Can Base Pair With mRNAmRNA is normally made using the noncoding strand of DNA as a template. Such mRNA is also known as sense RNA. If RNA is made using the coding strand as a template, it will be complementary in sequence to mRNA and is known as antisense RNA. The sense and antisense strands of RNA can base pair and prevent translation. In this example, antisense RNA is targeting its own mRNA for regulation of gene expression. If this were a pathogen’s genome, then the infected cell’s own nucleases would recognize and cleave the dsRNA, as discussed in the next section.
3. Figure 20.02. Antisense RNA Regulates Bacterioferritin SynthesisThe bacterial chromosome contains genes for both bfr mRNA and antibfr RNA. If both RNA molecules are transcribed the antibfr RNA pairs with the bfr mRNA and prevents it from being translated. When iron is plentiful the antibfr gene is not expressed and only the bfr mRNA is produced. Under these conditions translation of the bfr mRNA to give bacterioferritin can take place.
4. Figure 20.03. Mechanism of RNA InterferenceIntruding double-stranded RNA (dsRNA) is recognized as foreign by RDE-4 and other proteins. Dicer cleaves the dsRNA into segments of 21 or 22 nucleotides with one or two base overhangs—pieces called short interfering RNA (siRNA). This is recognized by RDE-1, which recruits the RNA-induced silencing complex (RISC). The strands of the siRNA are separated during RISC activation. Finally, RISC cleaves target RNA that corresponds to the siRNA.
5. Figure 20.04. Micro RNAMicro RNA (miRNA) is made by processing a longer precursor that folds into a stem loop. Processing occurs in two steps using the nucleases Drosha and Dicer. After binding to miRISC and strand separation, one strand of the miRNA binds to the target mRNA and prevents translation.
6. Figure 20.05. Structure of DicerDicer has multiple domains including a dsRNA-binding domain to hold the target mRNA, a PAZ domain that binds to the 3′-nucleotide overhang on the target, and two RNase III domains that cut a 21–23 nucleotide siRNA.
7. Figure 20.06. Amplification of RNA Interference by RdRPAnomalous RNA generated by RISC-mediated cleavage is used as a template by RNA-dependent RNA polymerase (RdRP). This generates more dsRNA, which in turn is converted into more siRNA by Dicer.
8. Figure 20.07. Experimental Induction of RNA InterferenceRNA interference occurs when both the sense and antisense RNA of a gene are present and form dsRNA. Three constructs are shown that direct the synthesis of a dsRNA molecule. The first construct (A) has a sense region and an antisense region that base pair. A spacer separates the sense and antisense regions and forms a loop at the end of the hairpin. The double promoter construct (B) has a promoter that directs the transcription of the sense strand and another promoter for the antisense strand. The two resulting RNA molecules are complementary and form a dsRNA molecule. In (C), dsRNA is created from two separate genes and two separate promoters.
9. Figure 20.08. Timeline of Select CRISPR DiscoveriesFrom the accidental discovery in 1987 through the numerous applications and even clinical trials of 2017, CRISPR technology has advanced biotechnology for many organisms.
10. Figure 20.09. The CRISPR SystemAn overall model of CRISPR/Cas activity. During spacer acquisition and adaptation, sequence elements from invading nucleic acids become incorporated at the leader-proximal end of the CRISPR locus. Chosen invader sequences are found close to protospacer adjacent motifs (PAMs). In the expression stage, the locus is transcribed and processed into mature crRNAs containing an 8nt repeat tag (gray) and a single spacer unit (multiple colors). During the interference stage, the mature crRNAs associate with Cas proteins to promote the degradation of complementary nucleic acids (two separate effector complexes are used for DNA and RNA targets).(Credit: Terns MP and Terns RM (2011) CRISPR-based adaptive immune systems. Curr Op Microbiol 14: 321–327).
11. Figure 20.10. Class 1 Type I crRNP Effector ComplexCascade is a large, multiprotein effector complex that recognizes one DNA strand through the complementary base pairing with guide crRNA and then Cas3 nuclease introduces a single strand break on the opposite strand.
12. Figure 20.11. Class 1 Type III Coupled ssRNA/ssDNA Degradation in TranscriptionInvading phage nucleic acid that is actively being transcribed is targeted with Class 1 type III systems. Here, the large effector complex recognizes ssDNA and resulting ssRNA in a transcription-dependent manner. The Cas10 and Cas7-like nucleases cleave both the ssDNA and ssRNA, respectively.
13. Figure 20.12. Class 2 Type II MechanismIn Class 2 systems, only single effector proteins are needed. The tracrRNA, RNase III, and Cas9 nuclease generate crRNA that combines with tracrRNA to generate a dual tracrRNA-crRNA for targeting. Double-stranded DNA complementary to the guide RNA is cleaved by Cas9 nuclease, creating a blunt-end.
14. Figure 20.13. Modification of Dual tracrRNA-crRNA to sgRNAA single-guide RNA (sgRNA) was experimentally generated by introducing an artificial linker between tracrRNA and crRNA. The sgRNA works as well as the wild type dual tracrRNA-crRNA for targeting and eliminates the need for two separate RNA molecules to be delivered to the experimental system.
15. Figure 20.14. Two Main Systems for Repairing Double-Stranded Breaks in DNANonhomologous end joining (NHEJ) and homology-directed repair (HDR) are two cellular pathways for repair of double-stranded breaks in DNA. NHEJ does not require sequence homology to repair the break and therefore can introduce insertions and deletions with more ease, but less predictability. HDR repairs the break by using a sequence with some homology to the area near the break. Experimentally, this allows DNA to be inserted into specific sites and is more predictable with fewer off-target effects.
16. Figure 20.15. Using CRISPR to Modulate Gene ExpressionAn edited Cas9 missing the nuclease activity can (A) prevent the binding or elongation of RNA polymerase at a promoter to repress transcription or (B) can be fused to a transcription factor to assist with the recruitment of RNA polymerase to the promoter to activate transcription.
17. Figure 20.16. Particle Bombardment of PlantsTarget DNA is coated onto metals, usually gold particles, which are then fired at plant cells using a particle gun. The coated metals pierce the cell wall and membrane and deliver the exogenous DNA into the cell. DNA is either expressed on the vector or may even integrate into the plant genome.
18. Figure 20.17. Agrobacterium-Mediated Delivery of Exogenous DNA Into PlantsAgrobacterium is a plant pathogen that transfers genetic information horizontally to the plant cell to induce tumor growth. The mechanism of transfer is exploited in genetic engineering to transfer target genes into plant cells.
19. Figure 20.18. CRISPR/Cas9-Mediated Conditional MutagenesisThe cas9 gene is under the control of a UAS. Gal4 in specific tissues and at a specific time point binds the UAS, expressing the cas9 gene into the nuclease. This enables tissue- and timing-specific editing using CRISPR. Knocking out genes involved in development at different developmental stages helps to identify gene function and regulatory networks.
20. Figure 20.19. CRISPR-mediated Targeting of Antibiotic Resistance GenesCRISPR systems are delivered into pathogenic bacteria via bacteriophage to eliminate the antibiotic resistance genes, often carried on plasmids. This can help prevent the spread of multidrug resistance in populations.
21. Figure 20.20. Principle of Gene DrivesGene drives do not adhere to standard Mendelian inheritance patterns and instead are inherited at a high rate. Without a gene drive and using Mendelian inheritance, only 50% of progeny would inherit a modified copy of a gene. As shown here, with a CRISPR-mediated gene drive for a single modified gene (red), the gene drive would copy itself and be transmitted at rates approaching 100%. Within a few generations, most of the organisms would have the modified gene.
22. Figure 20.21. Competitive Inhibition of Cas9 by AntiCRISPR ProteinsA bacteriophage carrying genes for antiCRISPR proteins infects a bacterial cell. As foreign DNA enters the cell, the phage-encoded antiCRISPR genes are expressed and competitively inhibit Cas9 from the bacterial cell’s natural CRISPR system. AntiCRISPR proteins bind to the amino acids that function in PAM recognition and catalytic activity of the nuclease domain. Binding of Cas9 by these small proteins prevents DNA binding and nuclease activity; thus, phage DNA is not destroyed.
23. Figure 20.22. Principle of ZFN Genome EditingZinc fingers recognize 3−4 nucleotides each. The binding of the zinc finger domain to DNA brings the fused FokI endonuclease in contact with a second FokI fused with zinc fingers recognizing the opposite DNA strand. The dimerization results in active nuclease that introduces a DSB that is repaired by either NHEJ or HDR.
24. Figure 20.23. Principle of TALEN Genome EditingTAL effectors contain sets of highly conserved 33−35 amino acid repeating sequences that differ by two amino acids, called the repeat-variable diresidues (RVDs) or hypervariable diresidues. These two amino acids determine which nucleotide base is recognized. For example, NI recognizes A, NG recognizes T, NN recognizes G, and HD recognizes C. The TALE DNA binding domain is flanked by both N- and C-terminal regions (not shown) and fused to an inactive FokI domain. Two FokI domains must associate to become active and introduce a DSB in the DNA. The TALE regions may be engineered to recognize different sequences on complementary DNA strands. Overall, each TALEN recognizes 16−24 bp target sequences.
25. Figure 20.24. Peptide Nucleic AcidPeptide nucleic acids have a modified amino acid backbone instead of a sugar-phosphate backbone present in polynucleotides. Protruding bases hydrogen bond with complementary bases. PNAs can form hydrogen bonds with DNA or RNA and are not usually recognized by nucleases.
26. Figure 20.25. Principle of PNA Genome EditingPNAs do not contain nucleases, but instead function by invading the double-stranded DNA and generating a triplex of PNA/DNA/PNA, that is then corrected by HDR or nucleotide excision repair. Experimental co-delivery of exogenous DNA leads to incorporation during the repair pathway and subsequent genome editing.