Topic 9 RNA Structure And Varieties

Question 1

What are the diverse biological functions of RNA and what types of rna do these do these

Answer 1

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

Question 2

Differences in RNA and DNA

Answer 2

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’

Question 3

Why would RNA have a 2’ OH if it makes it unstable

Answer 3

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

Question 4

Why would RNA make A PAIR with U and not T

What is the advantage

Answer 4

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

Question 5

Is it true that rna is a single stranded structure

Answer 5

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

Question 6

What similar structure can rna fold into

What are its characteristics

Answer 6

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

Question 7

What are the double helical characteristics of RNA

Answer 7

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)

Question 8

Explain the tetraloop structure of RNA

What interactions stablize it

Answer 8

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

Question 9

Explain the pseudoknot structure of RNA

Answer 9

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

Question 10

What are the structures that rna can make

Answer 10

Pseudoknot

Tetraloop

Question 11

How is dsDNA diff from dsRNA

what makes it a form and not b form helix

Answer 11

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

Question 12

Explain the how rna acts as thermosensor for virulence gene expression

Answer 12

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

Question 13

How can secondary structure rna (dsRNA) fold into tertiary structure

Answer 13

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

Question 14

What is the U:A:U base triple structure

What is special about what happens

Answer 14

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

Question 15

What are the implications of rna having the 3D structure

Answer 15

Can make an RNA switch

Aptamers

RNA fluorophore complexes

Ribozymes

Question 16

Explain the RNA switch

Answer 16

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

Question 17

Why is it a 5-10% chance of the gag-pol in the rna switch to happen

What does this mean

Answer 17

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,

Question 18

Why is the protonation of the adenine important in the riboswitch

Answer 18

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

Question 19

What is the benefit to having the gag (structural gene) and the pol (enzymatic gene) in the same mRNA transcript

Answer 19

1. If just one mRNA transcript with two genes you can save making an extra promoter region which is better
2. 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

Question 20

What are aptamers

Answer 20

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

Question 21

How are aptamers identified

Answer 21

By selecting from a large random pool of nucleotide or peptide sequences

Question 22

What is aptamer mediated precision therapy

Answer 22

Trying to find the best aptamer to be used in cancer therapy to trigger apoptosis of the cancerous cell

Question 23

What is used to make aptamers

Answer 23

SELEX

(Systematic evolution of ligands by exponential enrichment)

Question 24

What is SELEX

Answer 24

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

Question 25

Explain how SELEX can be used to make rna fluorophore complexes

Explain how it can help in metabolite structures and why

Answer 25

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

Question 26

What are the conditions of being an enzyme

What is special about rna

Answer 26

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

Question 27

What is a ribozyme

Ribozymes structure

Answer 27

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)

Question 28

How do ribozymes work

Answer 28

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

Question 29

Overall Whag happens to the rna if high pH

Answer 29

The ribozymes activity happens which caused rna degradation

Question 30

What is RNase P

Answer 30

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)

Question 31

What’s another example of a ribozyme

Answer 31

The group 1 introns

Question 32

What does RNAase P look like

Answer 32

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

Question 33

Explain how the RNase P works

Answer 33

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

Question 34

What is the advantage of rna over dna

Answer 34

RNA allows for greater range of base pairing than dna through non watson crick base pairing