RNA (lectures 6-9) Flashcards
What is the process by which DNA is copied into RNA by a template-directed polymerization?
Transcription
RNA transcripts can be decoded to generate cellular proteins in what process?
Translation
Describe the difference in strands between RNA & DNA
RNA - single-stranded
DNA - double-stranded
What is the chemical difference between RNA & DNA?
There is an absence of a hydroxyl group in deoxyribose (DNA), compared to ribose (RNA).
What is the difference between a nucleotide & a nucleoside?
Nucleotide = sugar + base + phosphate
Nucleoside = sugar + base
Describe what type of phosphate is present (moving away from sugar)
- Alpha
- Beta
- Gamma
What’s the difference between the nucleotides in RNA vs DNA?
RNA - uracil
DNA - thymine
What are RNA stem-loops?
Short helices which form as a result of intramolecular base-pairing
What is canonical (Watson-Crick) pairing?
GC & AT bonding
What is non-canonical pairing?
Pairing between nucleotides that aren’t regular (e.g. not GC, AT, AU pairings)
What non-canonical pairing is common in RNA?
G-U pairing
What is the A-minor motif?
Minor grooves in 2 consecutive A residues lead to interaction with adjacent GC base pairs.
What molecule contains a short RNA/DNA heteroduplex?
RNAP (RNA polymerase) - their active site
Where is RNA polymerase targeted on genes?
Promoter regions
When does transcription cease?
When the polymerase reaches the terminator region - it is released from the DNA.
Describe the core enzyme of the RNAP (RNA polymerase) of E. coli
It is a protein complex containing 5 subunits
What is the purpose of the sigma factor binding to RNA polymerase?
To provide specificity to the RNAP for the gene promoter.
- RNAPs can initiate transcription without primer activity.
Where are sigma factors found?
Bacterial cells
How many nuclear RNA polymerases do eukaryotic cells have?
3
Is the core structure of RNAPs conserved throughout eukaryotic cells?
YES
How many RNA polymerases do prokaryotic cells have?
1
How many nuclear RNA polymerases do eukaryotic cells have?
3
What is the 2 most used polymerases in eukaryotes?
RNA polymerase 1 & RNA polymerase 2
(Both 40% of RNAP activity each)
What is the role of RNAP 1?
Production of rRNA
What is the role of RNAP 2?
Production of mRNA, non-coding RNAs
What is the role of RNAP 3?
Production of tRNA
What is the purpose of General transcription factors (GTFs)?
Allow assembly of RNAP onto gene promoters in eukaryotic cells
Where are A/T-rich TATA boxes found?
On eukaryotic promoters
After the TATA box binding protein (TBP) binds the promoter, what is then formed following a series of interactions?
Preinitiation complex (PIC)
What is the benefit of transcription being coupled with translation in E. coli?
Allows rapid gene expression to occur.
What is the benefit of transcription & translation being uncoupled?
It is spatially separated allowing the RNA to be processed in the nucleus.
What is transcription?
Production of RNA
What is translation?
Synthesis of protein through reading of RNA
What are the 3 steps involved in the processing of eukaryotic mRNAs?
- capping of the 5’ end
- removal of introns (pre-mRNA splicing
- 3’ end processing (cleaving & polyadenylation)
What is polyadenylation?
Addition of poly-A tail makes the RNA molecule more stable & prevents its degradation
What is the name of the cap on eukaryotic mRNA?
m7G cap
Where is the m7G cap found?
yeast, animals & higher plants
What nucleotide modification takes place through the addition of the m7G cap?
guanosine nucleotide added to the 5’ end of RNA pol 2 transcripts
How is the cap linked to the transcript?
5’-5’ triphosphate linkage
Prokaryotes have polycistronic mRNA. What does this mean and what is its benefit?
This mean multiple, functionally related proteins can be created from a single mRNA. It is useful as it allows for a quick response.
Eukaryotes have monocistronic mRNA. What does this mean?
Each mRNA codes for a single polypeptide.
Why are genes in eukaryotic cells described as split genes?
As intron sequences within the mRNA are removed, leaving just the exon sequences
What allows pre-mRNA splicing to be accurate?
The recognition of spice site sequences
What 2 splice sites sequences are highly conserved (at the 5’ end & 3’ end)?
5’ - GU
3’ - AG
What nucleotide is a branchpoint in introns?
A
What is the name given to the complexes that carry out splicing?
Spliceosomes
What are active spliceosomes assembled from?
“snurps” (small nuclear RNPs [ribonuclear protein])
What are the 2 transesterification reactions?
- The 2’ hydroxyl group of the branchpoint adenosine attacks the 3’ phosphate of the 5’ exon.
- The 5’ - 2’ phosphodiester bond gives a looped lariat (lasso). - The 3’ hydroxyl group attacks the 5’ phosphate of the 3’ exon, releasing the lariat.
What has to be present in order for the ester bond to be broken & then recreated in the transesterification reactions during the catalytic mechanism of splicing?
Alcohol
What is the name given to enzymes with an RNA catalytic subunit?
Ribozymes
Describe the distribution (%) of the following Cellular RNA: mRNA, rRNA, tRNA - make up 90%
mRNA - 5%
rRNA - 75%
tRNA - 10%
(some small stable RNAs)
What are 3 reasons that mRNA is not clearly visible on the gel?
- cells transcribe many different mRNAs
- mRNAs have different sizes
- mRNA is generally unstable
What is an open reading frame (ORF)?
Sequences of DNA or RNA that can be translated into a protein.
What is the triplet (and amino acid) used as an initiation codon?
AUG - methionine
What does tRNA stand for?
transfer RNA
In what direction are codons read (letter & number)?
5’ - 3’
N’ - C’
Describe what is meant by charged tRNAs become ‘aminoacylated’
They become connected to an appropriate amino acid.
What type of bond forms between the aminoacyl & peptidyl groups, attached to adjacent tRNAs?
Peptide
Describe what has to occur for a tRNA molecule to become charged
An amino acid needs to become attached (aminoacylated).
Describe the process of in vitro translation (E. coli cell extract)
- isolate cell extract
- degrade mRNA with RNase
- inactivate RNase
- add RNA and amino acids
- precipitate protein & collect by filter binding
What complex formation is necessary for ribosome binding to occur?
ribosome/aa-tRNA/codon complex
How many ‘unique’ codons are there (codons that are the only triplet to code for a specific amino acid).
2:
AUG (start codon)
UGG (tryptophane)
What does it mean that the genetic code is degenerative (redundant)?
Amino acids are encoded by multiple codons
What are synonymous codons?
Codons that encode the same amino acid (tend to differ at the 3rd nucleotide of the codon)
What is the name of tRNAs that can become aminoacylated by synonymous codons?
isoacceptor tRNAs
What is the type of pairing which allows isoacceptor tRNA to become aminoacylated by synonymous codons?
wobble base pairing
What type of structure is given to tRNAs?
cloverleaf secondary structure (amino acid attached to the 3’ hydroxyl of the the 3’ terminal A nucleotide
What is the benefit of coaxical stacking which allow tRNAs to be folded into an L-shape?
Increases stability if helices are stacked together
What is needed to convert an amino acid + tRNA into an aminoacylated tRNA?
ATP
Ribosomes are made up of 2 unequal subunits. What are these 2 subunits (they are both the same)?
RNPs (ribonuclear proteins)
Does the codon bind to the small or large subunit of the ribosomes?
Small subunit
Does peptide bind formation occur on the small or large subunit of the ribosomes?
large subunit
How many tRNA binding sites are found on the small subunit of ribosomes?
3
How many tRNA molecules can bind to the 3 binding sites at one time?
2
What is the role of the small subunit in ribosomes?
Decoding codons
What is the role of the large subunit in ribosomes?
Peptide bond formation
What occurs at the PTC (peptidyltransferase centre)?
Bond formation
Describe the order at which a tRNA molecule moves through the 3 sites at the ribosomes
binds to A site, moves to P site, then when no longer charged, it moves on to the E (exit) site.
Where does rRNA transcription & rRNA processing occur?
Nucleoli
What is the benefit of the ribosomes only becoming function after leaving the nucleus?
Prevents the ribosomes from interacting with structures inside the nucleus
What joins the aminoacyl & peptidyl groups to the tRNA?
carboxylic ester linkages
Where are each of the 2 tRNA molecules bonded to at any one time at a ribosome?
- A&P sites - pre-translational state
- P&E sites - post-translational state
What elongation factor brings aminoacyl-tRNA to the ribosome?
EF1A (EF-Tu)
What elongation factor is required for translation?
EF2 (EFG in prokaryotes)
What are EF1A & EF2?
GTPases (elongation factors)
How many GTP molecules are hydrolyzed per incorporated amino acid?
2
To what site (on the ribosome) is the start codon directed to?
P site
What recognizes the start codon in eukaryotes?
Elongator methionyl-tRNAs
What recognizes the start codon in prokaryotes?
The SHINE-DALGARNO (SD) SEQUENCE
What is the Kozak sequence?
Translation initiation enhancer sequence?
What is the termination factor in eukaryotic cells?
eRF1
