RNA Flashcards
How is RNA formed?
RNA is synthesised by polymerases in the nucleus - specifically the nucleolus
Pol I - ribosomal RNAs
Pol II - all messenger RNA and small regulatory and nuclear RNAs
Pol III - transfer RNAs and small ribosomal RNA
All RNA processes lead up to translation
But everything requires RNA even the processes being used to make further RNA - largely interconnected
What is the biochemistry of RNA?
Ribose, nitrogenous base (A, U, G, C) and a phosphate group
Ribose has a hydroxyl group at C2 = less stable in alkaline conditions
ssRNA is produced but they can form secondary structures via base pairing and base stacking
How can ssRNA form secondary structures?
ssRNA adopts partially double-stranded, branched structure
RNA (double stranded regions) adopts A conformation – restriction enzymes do not recognize it
The addition of divalent cations promotes RNA folding - phosphate charge neutralization and formation of salt bridges
Looping facilitates a tertiary structure
How is rRNA processed in prokaryotes?
Ecoli has 3 types of rRNA - 5S, 16S and 23S
RNases cleave pre-rRNA to produce rRNA
How is rRNA processed in eukaryotes?
Takes place in the nucleolus, continuing through the cytosol, back to the nucleoplasm and then cytosol again
snoRNAs (small nucleolar) direct the methylation of rRNA
They direct a protein complex with a methyltransferase to the methylation site
What is significant about some eukaryotic rRNA?
Some are self-splicing e.g. tetrahymena pre-rRNA
Catalytic ability of RNA = ribozyme
Self splicing RNAs are known as group I introns
Group II introns - form a lariat intermediate
Describe riobozymes?
RNA-based enzymes = ribozymes
e.g. ribosome
Ribozymes exhibit all the same feature as protein enzymes: multiple turnovers, Michaelis-Meuten steady state kinetics
BUT
Mg2+ is essential - stuctural stability and catalytic activity
Hydrophobic pocket precisely complementary in shape to product
extensive stacking and hydrogen bonding
Give an example of a ribozyme?
Hammerhead ribozyme - they are small self-cleaving RNAs
Discovered in viroids and satellite RNAs of plant viruses
Catalyse a specific phosphodiester bond isomerisation reaction in the course of rolling-circle replication (in plant viruses)
What is the theroy of the RNA world?
As RNA can self replicate and evolve
RNA preceeded DNA as the first genomic, self replicating molecule DNA arose from RNA
SELEX - Systematic Evolution of Ligands with EXponential enrichment
This is forced molecular evolution of RNA molecules with desired properties in vitro
How is tRNA processed?
The tRNA genes are transcribed to give pre-tRNA molecules
The RNA is processed but this is NOT splicing
RNase P - ribozyme that processes pre-tRNA (uses Mg2+)
It removes the 5’ end leader sequence
= mature tRNA (cloverleaf-shaped secondary structure)
What do many eukaryotic pre-tRNAs have?
Eukaryotic pre-tRNA molecules can contain a short intron immediately 3’ to the anticodon
The intron is excised by an ATP-dependent splicing endonuclease
exons are then joined by an RNA ligase
What are the dominant features of the cloverleaf secondary structure of tRNA?
- 5’ terminal phosphate
- Acceptor stem - accepts the amino acid
- D-arm
- Anticodon arm
- T arm
- A 3′ CCA sequence with a free 3′-OH group
They are rich in modified bases
Describe the tertiary structure of tRNA?
L-shaped
Where the acceptor and T stems form one leg and the D and anticodon stems form the other
Each leg is 60 Å long
The anticodon and amino acceptor sites are at opposite ends
It is maintained by extensive stacking interactions and base pairing within/between its helical stems
What attaches amino acids to tRNAs?
Aminoacyl-tRNA synthetases
- The amino acid is “activated” by a reaction with ATP to form an aminoacyl–adenylate (all but 3 aa do this without the presence of tRNA)
- The mixed anhydride then reacts with tRNA to form the aa–tRNA
Amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + PPi
The aa is activated and the tRNA is charged as the aa-tRNA complex is a high-energy compound
What are the two classes of aminoacyl-tRNA synthetases?
Class I
Contains 2 conserved motifs (Rossmann fold) that bind ATP
Recognises the anticodon loop
Aminoacylate 2’OH on tRNA
Amino acid targets are larger/hydrophobic
Class II Contain 3 conserved motifs Don't recognise the anticodon loop Aminoacylate 3'OH on tRNA Amino acid targets are smaller/less hydrophobic
