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19 Cards in this Set
- Front
- Back
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Prokaryotic sRNAs
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small RNAs (80-110 nucleotides)- They bind to target mRNAs with assistance of Hfq (sRNAs aren't perfect matches with targets and Hfq helps stabilize.) They bind the mRNA and control the availability of ribosome binding sites.
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RybB RNA
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targets several mRNAs and triggers their destruction because of the double strand stretch of heteroduplex that informed upon pairing is recognized as a substrate of RNase E. most of its target encode for iron storage proteins (it regulates iron levels in the cell) It inhibits the transcription of sigma e.
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What is a riboswitch?
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mRNA that have complex 2nd structure in their 5' untranslated region. They control gene expression in response to changes in the concentrations of small molecules. They are made up of an aptamer and an expression platform. The aptamer binds small molecule regulator that changes their conformation and thus the conformation of the RNA in the downstream expression platform. It causes the formation of a stem loop transcriptional terminator.
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Riboswitch + B. Subtilis
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many genes are involved for a the use of methionine. They have 200 nucleotide untranslated area that acts as a riboswitch (SAM). It binds to the optima and stabilizes the structure ( which includes the RBS; called sequestering the RBS) preventing the polymerase from transcribing. This is also know as ATTENUATION.
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Riboswitch + tRNA
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also can respond to uncharged tRNAs (without attachments) binding of the uncharged tRNA allows for the RNA secondary structure to be stabilized and the tRNA synthetase gene can proceed. (there are many other things that Riboswitch can respond to)
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Controlling the Trp Operon
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Controlled by a leader sequence in the mRNA. Only expressed when tryptophan is limiting. the Trp operon mRNA encodes a leader peptide with 2 Trp. residues in a row. At low Trp, the ribosome stalls at this site, the ribosome stalls at this site and then allows transcription to continue. High Trp, the ribosome continues through the leader and the transcription attenuator forms preventing transcription. ONLY IN PROKARYOTES Because of simultaneous transcription and translation.
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CRISPR
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The prokaryotic immune system. (Clustered regularly interspaced short palindromic repeats) the repeated squences are ~30bp long. They are closely associated with Cas genes (CRISPR associated) Protospacers are added near the leader sequence and next to a protospace adjacent motif (PAM.) Some Cas proteins are required to make a protospacer. The new spacer is added to the proximal end of the leader sequence. The spacer increased resistance to the phage that supplied it.
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Cascade
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a complex of Cas proteins that cleave pre-crRNA into CRISPRs. The Cascade is guided by the spacer RNA to the foreign DNA where it is cleaved. This is found in E. Coli. P. furiosus has a similar enzyme but it targets foreign RNA instead.
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Why is everyone so excited about CRISPR tech?
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CRISPR can be used to disrupt specific deleterious genes that cause disease and CRISPR can be used to correct genetic defects using homologous recombination.
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How does RNA interference control gene expression?
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act by inhibiting translation of the mRNA, destruction of mRNA or even by transcriptional silencing of the promoter that directs expression of that mRNA.
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Generation of siRNAs and miRNAs and their mode of action
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dsRNAs (introduced into cell from virus) are processed to siRNAs and pre-miRNAs are processed to make miRNAs by an enzyme Dicer (it's similar to RNA III pol). Drosha is also used in the generation of pre-miRNAs. the combo of si/miRNAs direct RNA-induced silencing complex (RISC) to repress genes in three ways: inhibiting translation, destruction of mRNA, transcriptional silencing of promoter, or destruction of mRNA. Note: siRNAs can recruit an RNA-dependent RNA polymerase to produces more dsRNA for Dicer to make more siRNA
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Amplification of RNAi
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Amplification is why RNAi is so sensitive. only a few sirens are needed to silence the target mRNA. Amplification also allows for siRNA propagation to occur in daughter cells. The siRNA-RISC complex can recruit an enzyme (RNA dependent RNA polymerase to targeted RNA and the siRNA acts as a primer for the enzyme to take the target and make more dsRNA. Dicer then takes the dsRNA and make more siRNAs
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miRNAs... how the eff do they form...
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are encoded in the genome as segments of longer transcripts. They are generated by two RNA cleavage reaction. The first is to separate the stem loop and then make the mature miRNAs. Both sides of the stem loop can give rise to functional miRNAs. Drosha and dicer are both needed to make a mature miRNA. the miRNAs can be found in any part of the mRNA.
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Drosha... what does it exactly do again....
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Also a member of RNase III family,it makes two cleavages that cut the stem-loop region of the pre-miRNA. It does this in the nucleus. it binds with DGCR8 to form the micro-processor complex. Drosha cleaves 11bps away from the dsRNA-ssRNA junction. Once this has occurred it is exported into the cytoplasm for further cleavage.
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How does Dicer help in the formation of miRNA
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Dicer cleaves 22 nucleotides away from the end of the miRNA. Dicer is actually three modules: two RNAse III domains and a dsRNA-binding domain called PAZ domain. Each RNase is responsible for cutting one strand of RNA. The distance between the PAZ domain and the dicer determines the 22 nucleotide cut out
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RNA-induced silencing (RISC) complex
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The Dicer generates the sequence that becomes the guide RNA (what gives the RISC its specificity and guides it to the target.) the central component is a protein called Argonaute, often an RNA-cleaving enzyme (but not all are, some repress translation.)
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Mechanisms of siRNA vs miRNA
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siRNA match their mRNA targets exactly and cleave them.
miRNA do not match their targets exactly, therefore they generally inhibit translation |
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Lin-4 miRNA binds within the 3'-UTR of its target gene lin-14 gene
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In, C. elegans, Lin-14 is a transcription regulator. It controls the temporal expression of genes during development. Lin-4 miRNA blocks Lin-14 translation, contributing to developmental temporal control.
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cis vs trans genes
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cis acts on its self while trans acts on others
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