Dr Mitchell Flashcards
What is RNA bound to protein in?
Ribonucleoproteins (RNP)
What are the differences between DNA and RNA helices?
The major groove of RNA is deeper and narrower than that of B-form DNA
What are the unusual base interactions within RNA?
- Non-canonical base-pairs
- Base-triples
- Tetraloops RNAs
What are the tertiary structure motifs in RNA?
- A pseudoknot structure
- The A-minor motif
What are the percentages of mRNA, rRNA and tRNA in the cellular RNA profile?
5%, 75% and 10%
How does rRNA differ from its primary transcripts?
The sections are separated by extra genetic information which is removed, leaving separate shorter mature RNAs
How does tRNA differ from its primary transcripts?
Its transcript has extra genetic information either side which is removed to live a single shorter mature RNA
How does mRNA differ from its primary transcripts?
The sections are separated by extra genetic information which is removed and polyadenylation occurs, leaving a single shorter mature RNA
To which end of the RNA are new NTPs added?
The 3’ end
What region of DNA is the Pribnow box?
-10
What are the 5 subunits in the core RNA polymerase of E.coli?
a, a, B, B’ and w
What is the function of the beta subunits in the core RNA polymerase of E.coli?
Catalytic activity
What is the function of the alpha subunits in the core RNA polymerase of E.coli?
Binds transcription factors
What is the function of the omega subunit in the core RNA polymerase of E.coli?
Assembly and stability
What are the functions of the sigma factor in the RNA polymerase holoenzyme?
- Increases affinity for promoters
- Decreases nonspecific DNA binding
How does transcription initiation occur in E.coli?
- Sigma factor subunit in transcription bubble scans for promoter
- Promoter binds and DNA melts
- Primer synthesised (start of mRNA)
- When RNA primer is 18 nucleotides long, the sigma factor is released and NusA protein binds
What are the intrinsic terminators in E.coli?
- RNA stem-loop structure
- G-C rich (stable) region at stem base
- 3’ U-rich tail
What is the method of rho-independent transcription termination in E.coli?
- The polymerase pauses at the hairpin structure of intrinsic terminator
- rU-dA base-pairs destabilise the RNA-DNA hybrid within the polymerase
What is the method of rho-dependent transcription termination in E.coli?
- rho is an ATP-dependent RNA helicase
- rho binds to C-rich terminator sequences within the RNA and then catches up with the polymerase
- rho uses its helicase activity to unwind the RNA/DNA base-pairing
What are the functions of the three RNA polymerases in eukaryotic cells?
pol I -rRNA
pol II - mRNA
po III - 5S, tRNA
What is unique about the catalytic subunit of pol II in eukaryotic cells?
It has an extended C-terminal domain
What is the method of transcription initiation in eukaryotes?
- Preinitiation complex assembled at pol II promoters
- TATA box binding protein in TFIID binds to the DNA followed by TFIIB
- pol II binds to the DNA with TFIIF
- TFIIE and TFIIH bind
- The helicase activity of TFIIH melts the DNA duplex, allowing priming
What is the torpedo model of transcription termination in eukaryotes?
- Transcript cleaved by cleavage/polyadenylation complex
- Xrn2 breaks downstream fragment
- Polymerase displaced
What are the three types of mRNA processing?
- Capping of the 5’ end
- Removal of introns (splicing)
- Polyadenylation of the 3’ end
How does RNA processing occur in eukaryotes?
The C-terminal domain on pol II is differentially phosphorylated during transcription, allowing coupling of transcription with RNA processing events
What is the structure of the mRNA cap in eukaryotes?
On the 5’ end. Two nucleotides bound 5’ to 5’ so can’t be hydrolysed
What is the method of 3’ end processing in eukaryotic mRNAs?
- 3’ end formed by coupled cleavage and polyadenylation
- poly(A) polymerase adds nontemplated adenylate residues to the 3’ end
- Cleavage and polyadenylation occurs 3’ of the consensus sequence AAUAAA
How are intronic and exonic sequences distinguished?
Through the recognition of highly conserved splice site sequences
How are spliceosomes formed?
They are assembled and disassembled from snurps (small nuclear RNPs)
What is the mechanism of splicing?
- Involves 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
- The generated 3’ hydroxyl group attacks the 5’ phosphate of the 3’ eon, releasing the lariat
What are the possible noncanonical base-pairings?
- A modified G nucleotide at the first position of the anticodon can recognise codons ending with C or U
- Inosine base-pairs with A, C or U
What are the 21st and 22nd amino acids?
- Selenocysteine can be formed from cysteine to make selenoproteins
- The codon UAG can encode pyrrolysine which is made from lysine in some archaeabacteria
What is the method of tRNA generation?
- RNase P generates the 5’ end by removing the additional genetic information to the dies
- The 3’ end is generated by endo- and/or exonucleases
- tRNA nucleotidyltransferase adds CCA to the 3’ end
- Any introns get removed
How are tRNAs folded into an L-shape?
- Coaxial stacking of helices and base -pairing between the ends of the TPsiC and D loops produce a flat, L-shaped molecule
- The anticodon loop and aminoacyl group are positioned at opposite ends of the molecule
How are tRNAs charged?
They are charged with the appropriate amino acid by aminoacyl-tRNA synthetases
What is the structure of a ribosome?
- Two subunits (Large and small) and 3 tRNA binding sites
- Codon/anticodon binding occurs on the small subunit at the decoding centre
- Peptide bond formation occurs on the large subunit at the peptidyltransferase centre
- Has a polypeptide exit tunnel in the large subunit
- Codon/anticodon binding occurs on the small subunit at the decoding centre
What is the method of ribosome synthesis in eukaryotes?
- Transcription and early assembly
- Modification
- Processing
- Transport out of the nucleolus to the cytoplasm and late assembly
What are the three ribosomal tRNA binding sites?
- The A (aminoacyl) site
- The P (peptidyl) site
- The E (exit) site
What is the method of the translation elongation cycle in ribosomes?
- Amino acids and mRNA at P and A
- Bond forms between them and P detaches
- mRNA moves up so first codon at E instead of P and amino acids move to P as another attaches to A
What is the role of GTPases in translation elongation?
- aa-tRNA brought to the ribosome by EF-Tu (A GTPase) and a GTP is hydrolysed
- EFG (Another GTPase) translocates the amino acid and a GTP is hydrolysed
How is the initiation codon recognised?
Using the prokaryotic initiator tRNA (tRNAMetf) in which the Met is formylated. In eukaryotes the initiator tRNA is not modified (Meti not Metf)
What is the method of translation initiation in E.coli?
- Initiator tRNA binds the P sire and is bound by initiation factor IF2 in a ternary complex
- 30S/mRNA/tRNA complex formed and 50S subunit associates
- Initiation complex formed by GTP hydrolysis by IF2
What is the method of translation initiation in eukaryotes?
- Ternary complex formed as initiator tRNA binds to GTP-charged eIF2
- Ternary complex binds 40S and other factors
- Complex binds 5’ end of mRNA through interactions with cytoplasmic cap binding complex, eIF4F
- Pre-initiation complex scans mRNA until it finds an AUG codon in the Kozak sequence
- GTP hydrolysis by eIF2 and initiation factors released
- 60S bound using eIF5
What is the method of translation termination?
- Stop codons recognised by protein termination factors
- Release factors 1 or 2 bind, triggering peptide hydrolysis
- RF3 hydrolyses GTP allowing release of release factor
- Initiation factors, ribosome recycling factor and EF-G dissociate the ribosome
