RNA Flashcards
(20 cards)
Explain the 2 effect of RNA’s 2’-OH on stability
The 2’-OH group makes RNA:
1. Chemically less stable than DNA
- 2’-O atom nucleophilic attack at P atom
- Structurally more stable than DNA
- in the double helical state due to its 2° structure
2 features that stabilizes the RNA
- 5’ cap of mRNA
- 7-methylguanosine is joined to the 5’-end of the mRNA in a 5’-5’ triphosphate linkage
- 5’capping protein protects the mRNA from degradation by 5’ exonuclease - Poly(A) tail of mRNA
- Added in a multistep process, initial transcript is cleaved at the poly(A) addition site by an endonuclease before poly(A) tail is added
- polyadenylate polymerase catalyzes the polyadenylation reaction
Types of base pairing in the secondary structure of the RNA
- Watson-Crick base pairing
- The commonly seen base pair (A-T) & (G-C) - Wobble base pairing
- Non-canonical pairing (G-U, I-C, I-U, I-A)
- Adds to variety of interactions and functionality
What are the 6 types of secondary structures of RNA?
- Double helix
- Double helical state of A-form double helix (2’-OH group makes it impossible to adopt B-form)
- Single-strand region - Bulge
- 1 or more unpaired nucleotide within a helical region - Internal loops
- Mismatch bases within a helical region
- more than 1 nucleotide
- Can be symmetric (same no. on each side) or asymmetric - Pseudoknots
- single stranded folds to a base-pair with a hairpin loop - Kissing hairpins
- Base pairing between 2 hairpin loops
- Can be intramolecular or intermolecular - Hairpin loop-bulge contact
- Base pairing between hairpin loop and a bulge
- Wobble (non-canonical Watson-Crick) base pairs
Primary structure of tRNA
- All organisms contain more than 20 types of tRNAs, at least one for each of the 20 a.a.
- The length of all tRNA varies from 60 to 95, but most have around 76 nt, with numerous modified bases
→ pseudouridine (Ψ), ribothymine(T) , and dihydrouridine (D) - For 76-nt tRNA, there’s 15 invariant positions and 8 semi-invariant positions that occur mostly in the loop regions. This plays a role in determining the 2° and 3° structure
- 1st anticodon allows wobble base pairing, which accommodates ambiguity in the 3rd base of the codons
Describe the structure of the features, including the 4 arms in the cloverleaf structure, of tRNA.
- 5’-terminal phosphate group
- All tRNAs end in the sequence CCA with a free 3’-OH group
- 4 arms:
1. Acceptor or amino acid arm - 7 bp stem that has a protruding 3’- single stranded element (CCA) to which an a.a. can be attached
- D arm
- 3- or 4- bp stem ending in a loop that contains multiple dihydrouridine (D) bases - T arm (TΨC)
- A 5-bp stem ending in a loop that contains the sequence TΨC - Anticodon arm
- A 5-bp stem ending in a 7-nt loop that contains anticodon, the triplet of bases that is complementary to the mRNA codon specifying the tRNA
Describe the tertiary structure of tRNA
The tertiary structure of tRNA is maintained through hydrogen bonding and base stacking interactions
Name the diverse functions of RNA
- Adaptor/cofactor
- For protein synthesis - Catalyst (ribozyme)
- Code for proteins (only mRNA)
- Genome (in viruses)
- Regulator
- Control gene expression
What is RNA as genomic material used for?
RNA act as template for DNA synthesis via reverse transcription
RNA act as template for RNA synthesis for RNA replication
Briefly describe the process of translation from mRNA to protein
mRNA carries the information from a gene and the ribosome (rRNA) binds to the mRNA.
tRNA brings the correct a.a. to the ribosome by matching the mRNA codon and the ribosome adds the corresponding a.a. to the growing protein chain (peptidyl transferase reaction)
This results in the translation of genetic message into protein
When and how did Thomas Cech demonstrate the ability of RNA as catalysts (rRNA)?
Splicing of introns in rRNA occurred even in the absence of functional protein enzymes as the rRNA has self-splicing activity.
Thomas Cech spliced rRNA in the absence of nuclear extract, and in the presence of denatured protein, both of which showed that splicing activity still retained.
What are ribozymes?
Ribozyme is the general term used to describe RNA with catalytic activity
Some ribozymes are direct against separate substrates, some have intramolecular (auto-catalytic) activity that limits the catalytic action to a single cycle
Briefly describe the Group I intron self-splicing mechanism (automated separation of coding exons and non-coding introns via self-splicing)
Splicing occurs via transesterification reaction
3’-OH of guanosine acts as a nucleophile, attacking the phosphate group of the nucleotide at the 5’ splice site, releasing the 5’ side of intron from the RNA
The 3’-OH of the 5’ exon becomes the nucleophile, attacking the phosphate group of nucleotide at 3’ end of the intron, completing the reaction
Structure and function of ribonuclease P (RNase P)
Ribonucleoprotein contains a single RNA molecule bound to a protein. This RNA possesses the ability to catalyze the cleavage of a pre-tRNA substrate.
The protein plays a peripheral role in stabilizing the catalytic RNA
Describe the structure of hammerhead ribozyme
The hammerhead structure is formed by pairing interactions between a substrate strand (green) and an ‘assister’ strand (red) which can be inter or intramolecular. Contains divalent metal ion, usually Mg²⁺
- Divalent metal ion at site A coordinates directly to the 2’ oxygen to promote the formation of a stronger nucleophile of its alkoxide form, 2’ O⁻
-Divalent metal ion at B site promotes the development of negative charge on the leaving 5’-oxygen (weakens the 5’ O-P bond)
- Originally discovered in plants, used as a basis to artificially engineer ribozymes
Name the purpose of non-coding RNAs and the 2 important classes of small non-coding RNA molecules
Non-coding RNA serves as regulatory/catalytic functions (tRNA/rRNA)
2 important classes:
- MicroRNA (miRNA): small, single stranded RNA (~20 nt)
- Small interfering RNA (siRNA): small, double stranded RNA (~20 nt)
Both binds to mRNA to trigger their degradation or silencing
MicroRNA & siRNA:
- ~18-20 nt
- Involved in post-___ gene silencing and RNA ___
Small RNAs:
- ~20-300 nt
- Involved in ___ of targets RNAs, synthesis of ___ DNA, ___ structure dynamics, ___ modulation, structural role, ___
Medium and large RNAs:
- ~ 300-10000 nt or more
- Involved in DNA ___, X-inactivation, DNA ___, gene ___, generation of other RNA classes (miRNA & small RNA)
MicroRNA & siRNA:
- ~18-20 nt
- Involved in post-transcriptional gene silencing and RNA interference
Small RNAs:
- ~20-300 nt
- Involved in modification of targets RNAs, synthesis of telomeric DNA, chromatin structure dynamics, transcription modulation, structural role, gametogenesis
Medium and large RNAs:
- ~ 300-10000 nt or more
- Involved in DNA imprinting, X-inactivation, DNA demethylation, gene transcription, generation of other RNA classes (miRNA & small RNA)
How RNA pull-down assay help to study the RNA-protein interaction?
RNA pull-down assay:
- RNA-protein is purified and incubated, then tagged with fluorescent protein/marker that retains the RNA sequence and protein structure
- Nuclear protein fraction is added and binds to the RNA sequence, unbound proteins are washed off
- Protein tag allows visualization of protein when imaged while nuclear protein are detected to determine the RNA sequence
How RNA immunoprecipitation and chIRP-MS help to study the RNA-protein interaction?
RNA immunoprecipitation
- antibodies specifically complementary to the protein of interest is added and binds to the protein (if present)
- Immunoprecipitation and purification of RNA allows sequencing or qPCR of the RNA
chIRP-MS (mass spectrometry)
- In-vitro cross-linking in cell occurs and biotinylated oligos are added
- Protein tag then added, unbound beads are washed and undergoes elution
- Mass spectrometry of RNA binding protein obtained and used to identify RNA-protein structure
Alternative splicing: different combinations of exons from the same gene are joined together, producing multiple mRNA transcripts.
State the advantages of alternative splicing.
- Different transcript isoform produced
- Encodes different proteins from a single gene
- Have different localization, translation, and decay
- Increases the complexity and diversity of eukaryotic transcriptome (cell lines/tissue differentiation)
