Topic 9 RNA Structure And Varieties Flashcards
What are the diverse biological functions of RNA and what types of rna do these do these
Information flow: mRNA and genomes of some viruses
Structural: rRNA for parts of the ribosome
Regulatory: can turn on/off gene translation, non coding RNA
Enzymatic activity: ribozymes (rna having enzymatic activity)
Shows rna very versatile
Differences in RNA and DNA
DNA:
double helix/stranded: more stable and not prone to Exonucleases
ATGC
Replicates
Deoxyribose: no 2’ OH, can’t be deprotonated
A pairs with T
RNA:
Singles strand: very labile and prone to exonuclease , unstable
AUGC
Doesn’t replicate
Ribose sugar: 2’ OH, meaning less stable because it can become deprotonated
A pairs with U (instead of T)
But
both have directionality whether it’s 5-3 or 3-5’
Why would RNA have a 2’ OH if it makes it unstable
The OH can serve as an electron acceptor/donor in h bonding
This allows for folding in RNA structure with a lot of twists and turns
Why would RNA make A PAIR with U and not T
What is the advantage
thymine has a methyl group that isn’t in uracil, meaning thymine is more stable than uracil
This is why DNA is more stable than RNA
The more stable thymine isn’t needed in rna since RNA is transient (short lived) and unstable.
it’s also energetically cheaper to make the uracil than thymine
Also
Since cytosine in DNA can be deaminated and turn into uracil
If DNA and RNA use the same molecules (U) there will be confusion of if that strand is dna or RNA
Is it true that rna is a single stranded structure
TRNA: It folds into a cloverleaf structure, meaning it isn’t just a single linear strecite
Viruses: when cells encounter a virus they make siRNA which is double stranded, not single stranded
MiRNA: no coding rna that’s folded back in a hairpin structure, no single stranded at some point, regulates gene expression
So RNA may not be a simple as just a single stranded structure
What similar structure can rna fold into
What are its characteristics
Folds like A-form DNA
major groove is narrow but deeper than b from
Minor is wide but shallower than b form
This structure allows rna to bind to protiens
What are the double helical characteristics of RNA
The RNA folds back on itself due to the complementarity in its sequence to make local regions of dsRNA that are similar to A form DNA
When incomplete complementary it forms bulge and internal loop structures
This secondary folding of the RNA as stem loops has special stabilizing properties to make rna less labile (easily altered)
Explain the tetraloop structure of RNA
What interactions stablize it
Six nucleotides in the rna make a loop by pairing 4 nucleotides
C(UUCG)G tetraloop
Stabilized by:
C pair G
U pairs G (non Watson crick base pairing, 2 h bonds instead of normal gc three)
C has h bonding with the phosphate backbone of uracil (makes them in close proximity)
There is base stacking between bases of opposite orientation (van der waal force) to further stabilize this loop structure
Explain the pseudoknot structure of RNA
After forming a hairpin structure there is a tail left of the rna
Base paring between non-contiguous complimentary sequences can make This tail fold up and base pair with the loop
Becomes a 3D structure in the form of a pseudoknot, can now be called a double stranded RNA
What are the structures that rna can make
Pseudoknot
Tetraloop
How is dsDNA diff from dsRNA
what makes it a form and not b form helix
The 2’ OH in rna make it a A form helix not b form
So dsRNA major groove is narrow and deep (no accessible to amino acid side chains
The dsRNA minor groove is wider and shallower (accessible to amino acid side chains of interacting protiens)
The protiens that interact can bind to the DSrna through the secondary structures like stem loop, hairpin and bulges structures
Explain the how rna acts as thermosensor for virulence gene expression
In listeria monocytogenes (virulent bacteria)
There is prfA transcription factor to turn on the virulence gene expression
To actually make the prfA there is the RNA transcript whcih has the prfA genes and also the RBS
The RBS is the ribosome binding site for translation initiation and is in the stem loop structure of the mRNA
At 30 degree, the secondary RNA structure loop blocks ribosome binding
At 37 degree, the melting of the secondary rna structure allows translation of prfA to start to express the virulence gene
This mRNA secondary structure is temp sensitive and can act as a thermo sensor to regulate the expression of the virulent gene
How can secondary structure rna (dsRNA) fold into tertiary structure
The unpaired free rna has rotational freedom which allows it to fold into the secondary structure to make tertiary structure
Non Watson crick interactions like G:U, unconventional interactions like U:A:U base triple interactions (2 h bonds each) or base backbone interactions
Let it go into the 3D structure instead of just 2D loop structure
What is the U:A:U base triple structure
What is special about what happens
A way for the rna to from a 3D structure which is important for it function
It’s unique to RNA
The bases that are hydrogen bonding using N-H as donors or N as acceptors can be protonated or deprotonated depending on the environment of the RNA
This can change how the nucleotides interact with each other and change the 3D structure
What are the implications of rna having the 3D structure
Can make an RNA switch
Aptamers
RNA fluorophore complexes
Ribozymes
Explain the RNA switch
murine leukaemia virus (MLV), a type of mRNA virus, causes leukaemia cancer in mice
In the mRNA sequence:
there is the Gag genes: encodes the structural protien
the pol gene: encodes the reverse transcriptase
These two mRNA segment are separated by the stop codon UAG
90-95% of time Gag genes are expressed 5-10% of time the gag and pol genes are expressed (Gag-pol)
Used NMR to sturdy the 3D structure of mRNA
Found that right after the UAG sequence, there is a pseudoknot structure which serves as a proton sensor
In this knot is an adenine residue that when deprotonated, changes knot structure and inactivates the knot, stops translation at the UAG
When protonated, knot active, allows translation to keep going
Why is it a 5-10% chance of the gag-pol in the rna switch to happen
What does this mean
Because under physiological conditions/pH there is a 5-10% chance of the adenine being protonated (and knot being active)
This is why the pseudoknot can serve as a proton sensor and as a riboswitch to either turn on or off the expression of the pol gene
When adenine protonated it can form the triple bases,
Why is the protonation of the adenine important in the riboswitch
When adenine protonated it can form the base triple to change the 3D stuructr of the rna
When the 3D structure is change the function of the RNA is change and in the case of the riboswitch is can turn on off off gene
What is the benefit to having the gag (structural gene) and the pol (enzymatic gene) in the same mRNA transcript
- If just one mRNA transcript with two genes you can save making an extra promoter region which is better
- When the protien is made you have the structural and enzymatic region of the protien
in viral particles it’s more efficient to package them both together than having two separate molecules to put together
What are aptamers
Means fitting parts
It’s either an oligonucleotide (short dna or rna strands) or peptide molecule (short protien strands) that binds to a specific targeting molecule
So the rna oligonulceotide can fold into a functional aptamer and bind to a bio marker , this bio marker can be bound or a target cell to bring the oligonulceotide to it
Most aptamers are synthetic and used for therapeutic purposes
How are aptamers identified
By selecting from a large random pool of nucleotide or peptide sequences
What is aptamer mediated precision therapy
Trying to find the best aptamer to be used in cancer therapy to trigger apoptosis of the cancerous cell
What is used to make aptamers
SELEX
(Systematic evolution of ligands by exponential enrichment)
What is SELEX
Synthetically make RNA molcules with random sequence
The aptamer is selected to have a specific affinity to a bio marker
The rna that bind to the bio marker are recovered
These selected candidates are amplified by PCR but also introduced to as many mutations as possible
This is because the introduction of many mutations alter the structure of the aptamer to increase its affinity to the bio marker
So while doing PCR they select again with the mutated aptamers to see if they bind with higher affinity to the bio marker, do multiple runs
Ultimately want to find a specific aptamer with very very high affinity to the bio marker
Explain how SELEX can be used to make rna fluorophore complexes
Explain how it can help in metabolite structures and why
The GFP was initially purified from jellyfish Aequorea Victoria , then used as a reporter in C. Elegans
But now SELEX can be used to make short rna oligonucleotide aptamers that mimic the GFP and bind to a fluorophore
This fluorophore with diff GFP mimicking aptamers it emits diff colours of light when lgiht shines on it
Metabolite structure:
When the Metabolite binds to the aptamer it’s stabilized the aptamer structure, this allows the metabolite aptamer complex to bind to a fluorophore and light up
Do this because they want to track where the metabolite trafficking is in the cell to see its function in the cell
Shows how rna can be used as a molecular sensor
What are the conditions of being an enzyme
What is special about rna
Needs to :
Bind to a substrate
Carry out a chemical reaction
Release the product
Repeats the process many times
RNA can also act as a biological catalyst/enzyme
What is a ribozyme
Ribozymes structure
An RNA molcule that is capable of catalyzing a chemical reaction and has enzymatic activity
Has some protien enzymatic functions:
Has an active site (the 2’ OH group)
a binding site for substrate
a binding site for a co-factor (ex. metal ion)
How do ribozymes work
Can do alkaline hydrolysis:
When ph is high, less protons, the 2’ oh of rna is deprotonated
The 2’ oh becomes a oxyanion (O-) nucleophile
This oxyanion attacks and cuts off the 3’ phosphate in its backbone
This cleaves the rna chain and makes a 2,3’ cyclic phosphate (both 2 and 3’ attached to phosphate now) on the ribose that just attacked and the other part of rna is release
This allows for self splicing in rna to slic out intron which is found in the group 1 introns of rna in certain bacteria
Overall Whag happens to the rna if high pH
The ribozymes activity happens which caused rna degradation
What is RNase P
The 1st discovered ribozyme
Acts as endonuclease to modify the the precursor RNA to turn into its mature tRNA form
Composed of two moieties:
RNA moietiy: catalyzes cleavage of the tRNA precursor to make the mature tRNA
Protien moiety: allows binding of the ribozyme to the RNA substrates (the immature tRNA)
What’s another example of a ribozyme
The group 1 introns
What does RNAase P look like
Called hammerhead molecules because look like hammer when bound to TRNA precursor
Has the top RNA moiety, has metal ions binding site to allow it to have better function to cleave tRNA, has the protien moeity that help bind to the tRNA precursor
Explain how the RNase P works
It cleaves a segment of tRNA from the 5’ end of the precursor tRNA by attacking the phosphorus centre
This matures the tRNA with a 3’ end and a protruding strand on the other side
Then it can be used in translation
What is the advantage of rna over dna
RNA allows for greater range of base pairing than dna through non watson crick base pairing
