BMB 400 chapter 20
How does 6S RNA control transcription in E.coli?
- 6S RNA accumulates at high levels in the stationary phase of bacterial growth - it binds to sigma70 subunit of the RNAP HOLOENZYME and down regulates transcription of many sigma70 promoters
What is bacterial protein Hfq and what does it do?
- An RNA chaperon in bacteria - it aids the binding of sRNA to its targets in bacteria - this is because complementarity between sRNA and the target mRNA is imperfect and short so the interaction is weak - Hfq also increases the stability of sRNA even before they are paired with their target
What is the mechanism of genome editing with wild type Cas9?
- Cas9 is programmed using a guide RNA specific to the location of editing - wild type Cas9 site specifically cleaves dsDNA which activates the double strand break repair machinery - in the absence of a homologous repair template, non-homologous end joining can result in insertions or deletions which disrupt the target sequence (used to knockout the target gene). - precise knock ins can be made by providing a homologous repair template that has the desired knock in gene, and making he homology directed repair pathway insert that gene into the double stranded break
What is the mechanism of how small RNAs inhibit expression?
- Dicer generates siRNA and miRNA from dsRNA - the RNA-induced silencing complex (RISC) complexes with a strand of small RNA to create a mature RISC complex that is specific for the RNA thats complementary to the small RNA - mature RISC targets RNA for degradation or translation inhibition
What is the importance of PAM for the activity of Cas9?
- a 2-5 nucleotide sequence of NGG must follow the target DNA for Cas9 to work. - this mechanism prevents Cas9 from destroying the target DNA that has been integrated into the CRISPR loci since PAM is only found in invading DNA
What is pri-miRNA?
- a long dsRNA - precursor to miRNA - carries a haripin shaped secondary structure
What is the mechanism of CRISPR?
- acquisition phase: invading DNA from viruses is cut into small fragments and incorporated into the CRISPR locus - crRNA biogenesis: CRISPR loci is transcribed and processed into crRNA. these act as a guide factor to target invading DNA - interference: trRNA/tracrRNA complexes with crRNA, and they both complex with Cas9 endonuclease. Cas9 cleaves foreign DNA that has a 20 nucleotide sequence thats complementary to the crRNA and is adjacent to the PAM sequence (protospacer associated motif)
When does X chromosome inactivation occur in placental mammals?
- at the 32-64 cell stage - the choice of which X chromosome to express is random - once the cell selects an X chromosome to inactivate, that same copy remains inactive in all the decedents of that cell
What is the mechanism of attenuation in the trp operon?
- before the first codon of the first trp gene there is a leader sequence that has 2 tryptophan codons - when tryptophan concentration is low, there is very little tryptophan charged tRNA (Trp-tRNA). When the ribosome reaches the trp codons it stalls because theres not Trp-tRNA - the stalled ribosome covers region 1 of mRNA allowing region 2 and region 3 to pair up in a hairpin - the pairing of region 2 and 3 prevents the formation of terminator hairpin (pairing between region 3 and 4), and the transcript is not attenuated resulting in the expression of the operon - if there is high trp concentrations and high trp-tRNA then the ribosome proceeds through the trp codons and blocks region 2, this allows regions 3 and 4 to pair up into the terminator, attenuating the transcript
What is another example of X linked inactivation?
- calico cats - one gene on the x chromosome influences fur color orange or black - if the cat is hetrozygous for that gene, the different orange and black fur patches show where one of the 2 X chromosomes is inactive
What are the requirements for attenuation in the trp operon?
- coupling of transcription and translation - a segment of DNA that can serve as a terminator because of its base paired secondary structure - an alternative secondary structure that does nnot allow the termination of transcription
How is miRNA generated?
- generated by 2 RNA cleavage reactions on pri-miRNA, through a a 2 step nucleolytic processing - first cleavage is done by enzyme Drosha/DGR8 and it liberates the stem-loop from pri-miRNA to create pre-miRNA - second cleavage generates the mature miRNA from pre-miRNA
What are the different genome editing protocols that can be used using Cas9?
- genome editing with wild type Cas9 - genome editing by double nicking with paired Cas9 - localization with defective Cas9
Why is it important for 6S RNA to down regulate sigma70 RNAP in the stationary phase?
- in the stationary phase, an alternative sigma called sigmaS/sigma38 is made - sigmaS/sigma38 competes with sigma70 for core polymerase - sigmaS/sigma38 directs RNAP to genes related to stress response that are needed to survive stationary phase - By down regulating sigma70, 6S RNA helps shift the expression to sigmaS/sigma38 promoters needed for survival
What is the mechanism of x chromosome inactivation?
- initiating regulator is long non-coding RNA called Xist, it is encoded in he X-inactivation center (Xic) on the chromosome to be inactive - Xist RNA coats the X chromosome that expressed it and recruits chromatin modifying and condensing factors
Why is using dsRNA to silence in mammalian cells difficult?
- injecting longer dsRNA into the cells is hard - the dsRNA will shut down all transcription in the cells
What is the mechanism of genome editing by double nicking with paired Cas9?
- mutated Cas9 makes a site specific single stranded nick in the target DNA instead of a double stranded cut - 2 sgRNA Cas9s can be used to introduce a staggered double stranded break which can undergo homology directed repair
What are examples of sRNA repressing and activating transcription?
- repression: sRNA can base pair with the ribosome binding site to inhibit the ribosome from binding - activation: in the event the ribosome binding site is covered by the mRNA creating a double stranded hairpin, sRNA can pair with the outside facing region of the hairpin, unmasking he ribosome binding site
What are ways RNAi is used as a tool for manipulating gene expression?
- researchers can knockdown gene expression - to silence genes in worms, express RNA in E.coli and then feed E.coli to worms and screen for phenotype
What are some differences between bacterial sRNAs and eukaryotic regulatory RNAs?
- sRNAs are larger (80-110 nucleotides) while regulatory RNAs in eukaryotes are short (21-30 nucleotides) - sRNAs are encoded in their final for by small genes instead of being formed by processing dsRNA precursors
How does slicing of RNA by RISC happen?
- short siRNA generated by dicer is incorporated into RISC - the dsRNA is denatured to provide the guide strand, resulting in the mature RISC complex that recognizes target RNA based on complementarity with the guide strand - Argonaute has a PAZ and RNase domain, PAZ domain recognizes the 3' end of the guide RNA - when the guide RNA base pairs with target RNA, the active site of the RNase domain is positioned appropriatly to cleave target RNA - cleavage happens in the middle of the dsRNA, between 10th and 11th nucleotides from the 5' end of the guide RNA
What are the main types of short RNAs in eukaryotes?
- small interfering RNAs(siRNA): made artificially or produced in vivo from dsRNA precursors - microRNA (miRNA): derived from precursor RNAs that are encoded by genes expressed in cells where those miRNAs ave specific regulatory functions
What are structural features of the pre-miRNA stem loop?
- stem is ~33bp in length - contains only a few mismatches - at the top of the stem is a loop of variable size, usually relatively large 10 nucleotides - Stem can be divided into 2 functional segments: ~11bp lower stem and ~22bp upper stem
What do small RNAs in bacteria regulate?
- the replication of plasmids - regulating gene expression, through regulating transcription or translation
How does centromeric gene silencing happen in S. pombe?
- the sequence elements of the centromere are transcribed from both strands, resulting in complementary transcripts that can hybridize to form dsRNA - dsRNA is acted on by dicer to generate siRNA - siRNA recruits an argonaute-containing RISC-like complex called RITS complex to the cenromeres - recruitment of RITS results in slicing of centromeric transcripts,which generates more siRNA - RITS also recruits proteins that locally modify nucleosomes by adding silencing makers
What does RdRP do?
- the siRNA-RISC complex recruits RdRP to the target RNA - using the siRNA as a primer, RdRP extends the siRNA to create a dsRNA molecule - Dicer is hen able to cut the new dsRNA into new siRNA, which in turn can go bind to new RISC complexes
How does Dicer cleave dsRNA?
- the top region of Dicer is made up of 2 RNase domains. Each domain has an active site and is responsible for cleaving one of the 2 strands of dsRNA - PAZ anchors the 3' end of dsRNA so that the RNase active site is ~22 nucleotides away from the base. The active domains then cut the loop region off
What is the mechanism of genome editing by defective Cas9?
- this variation of Cas9 does not have nuclease activity but can still bind DNA. - this variation of Cas9 can be used to target specific sequences in an region of the genome without cleavage - the Cas9 protein can be fused with other proteins like gene specific effectors (activators or repressors) to activate or repress nearby genes. - can also be used to visualize when fused to GFP
How do small RNAs inhibit target genes in eukaryotes?
- trigger the destruction of mRN, when the small RNA is highly complementary to target RNA (typically with siRNA) - inhibit translation, when he small RNA is is not very complementary to target RNA (typically with miRNA) - induce silencing chromatin modifications within the target gene
What is the typical length of siRNA and miRNA?
21-23 nucleotides long
What is the structure of Dicer?
3 modules: 2 RNase III domains and a dsRNA binding domain called the PAZ domain. a linker domain connects the 2
What is the length of pre-miRNA generated by Drosha?
65-70 nucleotides
What is an example of sRNA that controls transcription?
6S RNA in E.coli
What is the enzyme that catalyzes the the second RNA cleavage in miRNA production?
Dicer
What is the enzyme that catalyzes the first step of miRNA generation?
Drosha
What is the mechanism of the of the first step of the nucleolytic processing to generate miRNA?
Drosha and Pasha/DGCR8 make 2 cleavages that cut the stem loop (pre-miRNA) out of the primary transcript RNA (pri-miRNA)
What part of pre-miRNA does the PAZ domain of dicer interact with?
PAZ forms a binding pocket for the 3' end of the dsRNA
What are riboswitches?
RNA regulatory elements usually found within the 5' untranslated region of the genes they control
Does Drosha work with anything else in the processing of miRNA?
Yes, it works with an essential specificity subunit protein called Pasha or DGCR8 to form an active microprocessor complex
What is the central component of RISC?
an RNA cleaving enzyme called argonaute
Why cant attenuation happen in eukaryotes?
because transcription and translation are not coupled
What is an example of transcription silencing by chromatin modification mediated by small RNAs?
centromere gene silencing in fission yeast S.pombe
What is an RNAi method that can be used to silence mammalian cells?
expressing short hairpin RNAs (shRNAs) from DNA in the cells
What part of the transcript can pre-miRNA be found?
in any part: within coding regions, leader regions, introns, exons, in protein coding pre-mRNA, in non coding RNA
Where does Drosha reside in the cell?
in the nucleus and that is also where the fist cleavage event of miRNA takes place
What is a significant feature of how Drosha cleaves pre-miRNA?
it leaves a 2 nucleotide overhang on the 3' end of the dsRNA which is important for recognition by the second enzyme Dicer
How does 6S RNA bind to sigma70 RNAP?
it mimics the B-form of DNA with a structure that resembles a replication bubble
Does the regulation of the trp operon require any protein transcription factors?
no attenuation is enough and it does not require repressors or activators
Where does Drosha cleave the stem?
right in between the lower and upper stem leaving a 22bp dsRNA and the top loop
What controls the expression of the trp genes?
the Trp repressor repressed the trp operon in the presses on high concentrations of tryptophan
What is another factor that adds to the efficiency of RNAi?
the actions of RNA-dependent RNA polymerase (RdRP)
What is a guide RNA?
the active form of regulatory RNA, which is in single strand form and incorporated into a RISC complex
Where does the second miRNA cleavage take place?
the cytoplasm since that is where dicer is
Why does RNAP sometimes not complete the transcription of trp genes?
the decision to make a complete transcript is controlled by attenuation, most transcripts are terminated prematurely but attenuation is overcome when tryptophan levels are low
What made it possible to use the Cas9 system to edit DNA?
the development of synthetic single guide RNA (sgRNA) that acts as a combination of trRNA and crRNA and then programming Cas9 with sgRNA instead of trRNA and crRNA
What is dosage compensation?
the mechanism where the body able to silence one of the X chromosomes in females to ensure that genes on the X chromosome are not expressed at twice the level
How can the presence of miRNA be predicted?
the secondary RNA structure of pri-miRNA is very distinct and makes it possible to predict the presence of miRNAb based on the secondary structure fold of the RNA sequence
What is the structural requirement for the processing of pri-miRNA?
there has to be ssRNA flanking each side of the of the stem loop
How are siRNAs and miRNAs generated?
they are both generated from longer dsRNAs by the enzyme dicer
What is the function of CRISPR and Cas genes?
they are essential in adaptive immunity in bacteria and archaea, mainly to fight viral infections
When are the trp genes in E.coli expressed?
they are expressed efficiently only when tryptophan is low
What is the most common mechanism for how sRNA work?
they base pair with complementary sequences within a target mRNA. This can lead to the destruction of the mRNA, inhibiting its translation, or stimulation of translation
How do riboswitches regulate expression?
they regulate expression at both the transcription and translation levels through changes in the RNA secondary structure
What do riboswitches respond to when controlling gene expression?
they respond to changes in the concentration of small molecules
How effective is silencing by RNAi?
very efficient, a small amount of dsRNA is enough to induce near complete shutdown of target gene