Topic 9 (RNA Structure) Flashcards
What are the biological functions of RNA?
- information flow (mRNA)
- structural (rRNA)
- regulatory (non-coding RNA)
- enzymatic activity (ribozymes)
What is a unique feature of ssRNA structure?
It has rotational freedom and flexibility
How is RNA’s functional diversity comparable to protein’s?
Not as functionally diverse as proteins
What is the flow of genetic information (central dogma)?
DNA -> RNA -> protein
Why is RNA more vulnerable to exonucleases than DNA?
Doesn’t need as long as a half-life and can be transcribed again if necessary. DNA encodes RNA so it must be kept preserved
What are the structural features of RNA?
2’ OH and uses U instead of T, otherwise the same as DNA
Is RNA or DNA more stable at high pH? Why?
DNA is more stable because the 2’ OH in RNA gets deprotonated in high pH, which makes it less stable
What is the benefit of RNA having the 2’ OH?
It serves as an electron acceptor/donor, which allows RNA to fold into various structures
Why is T found in DNA rather than U?
T is more stable (DNA needs to be stable), it’s harder to synthesize (requires more energy), so it can’t be used in RNA, and U is detected by DNA repair machinery as a result of C deamination
Why is U used in RNA?
It costs less energy to synthesize, and RNA is synthesized many more times than DNA is within one cell’s life, so it’s more energetically favourable
Can RNA be double-stranded?
Yes, such as in siRNA and miRNA
What form of DNA can RNA fold into?
A-form DNA
What are the groove characteristics of A-form RNA?
Deep but narrow major groove, shallow but wide minor groove
What are the structures sometimes formed by dsRNA?
Bulges, internal loops, junctions, and hairpins
How does a bulge occur?
One base is unable to base pair on one strand, while all other adjacent bases pair regularly. For example, the top strand: 5’-GAC-3’, and the bottom strand: 3’-CG-5’, A in the top strand would bulge
How does an internal loop occur?
Incomplete complementarity between two RNA strands
What is the tetraloop structure? What is the associated sequence?
A complex structure in which RNA may take that involves non-Watson-Crick base pairing, base stacking, and hydrogen bonds with the backbone; C(UUCG)G
The first base in C(UUCG)G (tetraloop) base pairs with _________ and has base-stacking interactions with _________
Base 6 (G); base 2 (U)
The second base in C(UUCG)G (tetraloop) base pairs with _________ and has base-stacking interactions with _________
Base 5 (G); bases 1 (C) and 4 (C)
The third base in C(UUCG)G (tetraloop) base pairs with _________ and has base-stacking interactions with _________
None; the sugar of base 4 (C)
The fourth base in C(UUCG)G (tetraloop) base pairs/hydrogen bonds with _________ and has base-stacking interactions with _________
The P of base 2 (U); base 2 (U)
The fifth base in C(UUCG)G (tetraloop) base pairs with _________ and has base-stacking interactions with _________
Base 2 (U); base 6 (G)
The sixth base in C(UUCG)G (tetraloop) base pairs with _________ and has base-stacking interactions with _________
Base 1 (C); base 5 (G)
Where is non-Watson-Crick base pairing in the tetraloop structure?
Between base 2 (U) and 5 (G)
Where is hydrogen bonding between a base and the phosphate backbone in the tetraloop structure?
Base 4 (C) and phosphate of base 2 (U)
Where are the base-stacking interactions in the tetraloop structure?
Between bases 1 (C) and 2 (U), 2 (U) and 4 (C), 3 (U) and the sugar of base 4 (C), and 5 (G) and 6 (G)
What is unique about the base stacking of base 3 (U) in the tetraloop structure?
It is not pi-stacking
What is a pseudoknot?
An RNA structure that forms through base pairing between non-contiguous complementary sequences (unpaired bases in a hairpin may pair with other bases farther down the sequence)
What prevents RNA from adopting a B-form helix?
Presence of 2’ OH
Where do proteins interact with dsRNA? Why?
Minor groove; major groove is too deep and narrow, which decreases its accessibility to proteins, while the minor groove is shallow and wide, which increases its accessibility
What do proteins recognize when binding to dsRNA?
Secondary structures (hairpins, stem-loops, bulges, etc)
Describe how L. monocytogenes works at low temperatures
PfrA (TF) is unable to turn on the virulence gene because the ribosome binding site for its transcript is inaccessible due to the secondary RNA structure
Describe how L. monocytogenes works at high temperatures
The secondary structure of the RNA is melted, forming a bubble where the ribosome binding site is. The ribosome can bind and translates the PrfA protein to turn on the virulence gene
Describe regulation of translation using temperature
L. monocytogenes can turn on/off gene expression depending on high/low temperature, respectively
When is a U:A:U base triple formed?
Usually in ssRNA, which helps the folding of tertiary structures (like tRNA)
What is the structure of the MLV transcript?
5’-Gag-UAG-pseudoknot-Pol-3’
How does MLV control translational readthrough of its mRNA?
Through the use of a pseudoknot
Gag is translated _____% of the time in the MLV transcript
90-95%
Pol is translated _____% of the time in the MLV transcript
5-10%
Describe how the pseudoknot works in MLV
Deprotonated: 90-95% of the time. Adenine forms a pseudoknot structure in which it crosslinks with another part of the transcript. This terminates translation at the UAG codon (stop codon). Gag is translated
Protonated: 5-10% of the time. Adenine is protonated and does not form a pseudoknot, allowing the ribosome to read through the UAG codon and translate Gag-Pol
What is a riboswitch?
Another name for the function of the pseudoknot in MLV
What kind of protein is Gag?
A structural protein
What kind of protein is Pol?
A reverse transcriptase
What are the advantages of having 2 genes encoding structural and enzymatic proteins in tandem?
Only need one transcript for two genes that can be regulated independently
What is an aptamer?
An oligonucleotide/peptide molecule that binds to a specific targeting molecule
True/False? Aptamers are man-made
True
How does an aptamer work? What is an application of this?
Binds to a specific biomarker on the surface of the target cell; can be used to trigger apoptosis in cancer cells
What does SELEX stand for?
Systematic evolution of ligands by exponential enrichment
What is SELEX used for?
To create aptamers that can bind to specific cells
Describe how SELEX works
- Synthetic synthesis of RNA molecules
- Selection of RNAs that bind to a specific ligand (selected based on affinity for their ligand)
- Amplification by PCR and mutagenesis (progressively enrich the aptamers with a higher affinity by introducing mutations)
- Repeat steps 2-3
- Recovery of RNAs with desired affinity
SELEXS works similarly to _______ in immunity
B-cell antibody synthesis
What is GFP?
Green fluorescent protein used as a reporter gene in C. elegans
Describe how an RNA-fluorophore complex works
- A metabolite-sensing RNA binds a metabolite
- Metabolite binding reinforces RNA structure
- Fluorophore binds RNA complex due to reinforced structure
- Complex glows, mimics GFPs but allows for a greater range in colours
Can RNA be an enzyme?
Yes. For examples, ribozymes are able to catalyze reactions like protein enzymes can
What are the five processes an enzyme must be able to do?
- bind a substrate
- carry out a chemical reaction
- release the product
- remain unchanged by the reaction it catalyzes
- repeat the process many times
Describe the steps of alkaline hydrolysis
- 2’ OH becomes deprotonated at high pH
- Oxyanion attacks 3’ phosphate
- RNA chain breaks, producing a 2’ 3’ cyclic phosphate and an RNA strand with a 5’ OH
Alkaline hydrolysis is an example of:
RNA self-splicing/degradation as a result from acting as a ribozyme
What three things are ribozymes composed of?
- an active site
- a binding site for the substrate
- a binding site for the co-factor
What is RNase P?
The first ribozyme discovered that generates tRNAs from large precursor RNAs through endonuclease activity
What are RNase P’s two moieties?
RNA: catalyzes cleavage of tRNA precursor, contains co-factor (metal ions)
Protein: facilitates binding to its RNA substrates
What does the shape of RNase P resemble?
A hammer
What is the function of the co-factors in RNase P?
Metal ions allow RNase to have more active function to cleave tRNA precursor
What does RNase P cleave off of the tRNA precursor?
The 5’ end is cleaved shorter than the 3’ end, which allows the 3’ end to bind to amino acids
