3. Some bacteria possess a fascinating system for combatting viral infections ca
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3. Some bacteria possess a fascinating system for combatting viral infections called CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) The genetic elements of the CRISPR locus (region) of the bacterial chromosome are arranged like this: Intervening spacer DNA CAS genes Palindromic Repeats The spacer DNAs are actually sequences from viral genomes, that were copied and placed between the palindromic repeats during a viral infection. Through the action of the Cas enzymes, the viral sequences can be used to target viral RNA or DNA for destruction, as shown in the figure to the right. DNA sequences NA Or DNA from different viruses are retained and can be used as a defense against the viruses in the future. Palindromic DNA foildback loops Processing by spacer RNAs Targeting of Cas-enzyme complex to virus RNA or DNA Cas Enzymes Cleavage of virus DNA/RNA by Cas enzymes Cleaved RNAONA A. Which steps in the CRISPR mechanism are most similar to the Dicer and RISC steps of the iRNA process of eukaryotes? B. Describe three similarities between the functions or mechanisms of the RNAi and the CRISPR. (e.g. In both CRISPR and iRNA...") C. Other than the specific enzymes involved, identify three differences between the RNAi and the CRISPR mechanisms. (e.g., In CRISPR ..., whereas in RNA... D. Explain the function of the palindromic repeats in the CISPR mechanism?Explanation / Answer
A. In CRISPR, the complementary guide-RNA finds its matching sequence in DNA, unwinds it, followed by double stranded cleavage with CAS endonuclease. In iRNA, Dicer cleaves dsRNA into short dsRNA fragments (small interfering RNA) with help of its endoribonuclease activity. RISC uses siRNA as template for locating The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template to locate the target mRNA (which is to be silenced) and cleaves by a ribonuclease. crRNA formation with help of Cas in CRISPR highly resembles RISC process.
The cleavage of DNA in CRISPR and cleavage of mRNA in iRNA resembles each there as they are mediated by nucleases.
B. 1. Both use RNA as a complentary sequence for targeting DNA. Guide-RNA is used in CRISPR and small interfering RNA is used in iRNA. 2. Both use endonucleases for cutting DNA or RNA sequences in CRISPR or iRNA respectively. 3. Both affects the gene expression; CRISPR creates gene knock-out while iRNA creates knock-down.
C. 1. CRISPR is a naturally occurring prokaryotic immune defense against viruses and exogenous plasmid RNA while iRNA is the eukaryotic gene silencing method which regulates gene expression. 2. In CRISPR, endonuclease CAS binds and cleaves double stranded DNA detected by completary guide-RNA while in iRNA, double stranded small interfering RNA is used to locate the template mRNA which is cleaved by endoribonuclease. CRISPR uses double stranded DNA break repair mechanisms to edit the sequence while iRNA is not editing tool rather it just silences mRNA. 3. CRISPR creates knock-out of a gene since it introduces mutation (insertion/deletion) at DNA level parmanently disabling the particular gene while iRNA creates knock-down of a gene since it silences mRNA (not at DNA level) resulting in less or no expressions of gene.
D. Palindromic repeats are transcribed along with spacers followed by cleavage with Cas results in crRNA which is used to target the DNA sequence to be cleaved. Palindromic repeats form the tracer RNA component of crRNA tightly bound with CAS protein while spacers form the guide-RNA which is complementary to the target DNA sequence.
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