Topic 9 Flashcards
What are the 4 functions of RNA?
- Information flow
- mRNA, and genomes of some viruses - Structure and synthesis
- RNA (rRNA) components of ribosome - Regulatory
- Riboswitch (RNA can function as a switch for gene expression)
- non-coding RNAs - Enzymatic activity
- Ribozymes
True or false: RNA is very labile (unstable) and vulnerable to exonucleases
True
Is RNA or DNA more stable at a high pH? Why?
DNA
- Because at high pH, 2’OH on RNA is deprotonated, making RNA very unstable
Deprotonation of 2’-OH makes the oxygen more nucleophilic. This can lead to an intramolecular attack on the adjacent phosphate group, causing the RNA strand to break through a process called alkaline hydrolysis.
Why is Uracil found in RNA but not DNA?
Allows DNA repair machinery to detect cytosine deamination due to presence of Thymine instead of Uracil
Which is more stable: Thymine or Uracil?
- Explain why
Thymine is more stable due to methylation
RNA can fold back on itself to form local regions of dsRNA, similar to…
A-form DNA
- Major groove is still deep but narrower/compressed
- Minor groove is wider compared to B form
Some RNA stem-loops have…
Special stabilizing properties
Tetraloop RNA sequence and function
C(UUCG)G
- Stabilizes certain RNA secondary structures
In what three ways does an RNA Tetraloop stabilize the RNA loop?
Through special interactions:
1. Non-Watson-Crick base pairing (non-canonical G:U Base Pair)
2. Hydrogen bonding between a base and the phosphate backbone
3. Base-stacking (Van der Waals) between bases of opposite orientation
What are pseudoknots?
Structure formed by RNA base pairing between non-contiguous complementary sequences
The presence of 2’- OHs in RNA prevents RNA from…
Adopting a B-form helix
- Double-helical RNA resembles the A-form DNA
Describe the major and minor grooves of dsRNA
Major groove: Narrow and deep (not very accessible to amino acid side chains)
Minor groove: Wide and shallow (accessible to amino acid side chains)
How do proteins bind dsRNA?
By recognizing the secondary structures, such as hairpins, stem-loops or bulges
Describe the thermosensor for virulence gene expression in Listeria monocytogenes example for why secondary structures are important for gene regulation
prfA is a temperature-sensitive TF that turns on the virulence gene
- At 30 degrees C, secondary RNA structure masks ribosome accessibility
- At 37 degrees C, melting of the secondary RNA structure allows translation to begin
Summarize to the best of you ability everything learnt so far…
Secondary RNA can fold into…
- Explain how
Tertiary structure
- RNA has enormous rotational freedom in the backend of its non-base pairing regions -> secondary RNA structure can fold into complex tertiary structure
-unconventional interactions, such as U:A:U base triple or base-backbone interactions, help form these tertiary structures
What can change the 3D interactions in the tertiary structure of RNA?
Protonation/deprotonation of bases change the 3D interactions
Describe the structure of the murine leukemia virus (MLV) RNA
RNA is separated into two regions:
- Gag (structural proteins), expressed 90-95% of the time
- Pol (reverse transcriptase), Gag-Pol expressed 5-10% of the time
These regions are separated by a UAG stop codon.
Describe how the MLV RNA acts as a riboswitch
Pseudoknot structure forms after stop codon at Gal-Pol region
- Serves as a proton sensor that can turn Pol expression on or off
- When adenine in pseudoknot is deprotonated, this inactivates the ribosome to read through the stop codon
- When the adenine is protonated, this activates the readthrough of ribosomes through the stop codon
What are the advantages to having 2 genes encoding a structural and enzymatic protein in tandem? (2)
- Saves a promoter region (beneficial for small viruses)
- More efficient to have the proteins translated together
What is an aptamer? How is it identified?
An oligonucleotide or peptide molecule that binds to a specific targeting molecule
- Aptamer sequence forms 3D structure, which binds to a biomarker on a target cell.
- Usually identified by selecting from a large random pool of nucleotide or peptide sequences
What are aptamers useful for?
Allows for adapter-mediated precision therapy (targeting specific cells for therapy)
What is SELEX? (3 steps)
Systematic Evolution of Ligands by Exponential Enrichment (SELEX):
1. Synthetic synthesis of random RNA molecules of a specific length
2. Aptamers are selected based on specific property, e.g. affinity for a specific molecule
3. Recovery of RNAs with desired affinity OR amplification by PCR and mutagenesis
- Progressively enriching the tailor-made aptamers with higher affinity b/c by introducing mutations, the RNA structure may change in a way to increase its binding affinity to a biomarker
Cycle continues (usually do ~10 rounds of this to find an aptamer that is really specific to the biomarker, which is specific to a target cell)
RNA-fluorophore complexes mimick…
GFP from jellyfish Aequorea victoria that was first used as a reporter in C.elegans
How are RNA-flurophore complexes made? (3 steps)
- Aptamer with distinct secondary structure has space to bind metabolite
- Metabolite binds to aptamer which stabilizes the aptamer and allows for binding of the fluorophore
- Fluorescent complex formation, which can be used to track metabolites in the cell (metabolite sensor)
Define a ribozyme
An RNA molecule capable of catalyzing a chemical reaction
Describe alkaline hydrolysis and the role of the ribozyme in this process
- 2’ hydroxyl becomes deprotonated
- Resulting oxyanion attacks 3’ phosphate
- RNA chain breaks, producing a 2’, 3’ cyclic phosphate
Breakage of RNA chain is catalyzed by ribozyme
Ribozymes are composed of what 3 things?
- An active site
- A binding site for different substrates
- A binding site for a co-factor (e.g. metal ion; different ribozymes can bind different cofactors)
RNase P function
- The first ribozyme discovered
- An endonuclease that generates tRNA molecules from large, precursor RNAs
RNase P structure
Composed of 2 moieties:
- RNA moiety: catalyzes cleavage of tRNA precursor (allows for maturation of tRNA)
- Protein moiety: Facilitates binding to its RNA substrates by binding cofactors
What do the metal ions in RNase P allow for?
Allows RNase to have more active functions to cleave the tRNA
Specifically, RNase P cleaves…
A segment of tRNA from the 5’ end of a precursor
- Results in protruding 3’ end from tRNA -> amino acid binding site
In the MLV mRNA, where is the pseudoknot located?
Within the Pol RNA
