NAGE Flashcards
Which bases are purines and which are pyrimidines?
AG = purines CUT = pyrimidines
How many base pairs are there per helical turn and how wide is the double helix?
10 bp per helical turn
2nm
State two ways of separating the DNA strand.
Heat
Low salt concentration
What is the lowest level of packing of DNA?
Nucleosome – it is wrapped around a histone (positively charged) which strongly attract to the negatively charged backbone of DNA – 200bp – causes 7 fold condensing
What is the next level of packing of DNA?
30nm fibre
What two types of nucleotides are needed for DNA synthesis?
Oligonucleotide primer (RNA primer made by DNA primase) dNTPs (deoxynucleoside triphosphates)
Explain how the lagging strand is synthesised.
DNA polymerase can only add dNTPs to the 3’ end of an oligonucleotide primer.
The oligonucleotide primers attach and DNA polymerase moves in a 5’ to 3’ direction adding the dNTPs to the growing chain. When the newly synthesised strand reaches the oligonucleotide primer of the previous okazaki fragment, the DNA polymerase is removed. Ribonuclease removes the RNA primer and Repair DNA polymerase replaces the RNA with DNA. DNA ligase then joins the adjacent okazaki fragments.
Which three enzymes are involved in the joining of Okazaki fragments?
Ribonuclease, Repair DNA Polymerase, DNA ligase
Which strand of DNA is transcribed?
Anti-sense (it is anti-sense to the mRNA produced)
What are the roles of the three types of RNA polymerase?
I – transcribes rRNA genes
II – transcribes genes encoding proteins into mRNA
III – transcribes tRNA and 5S RNA genes
What is the role of gene promoter?
Transcription complex assembles at the gene promoter
What constitutes the basal transcription complex?
TFII (D, A, B, F, E, H, J) and RNA polymerase II
How do transcription factors affect gene expression?
By modifying histones
Hyperacetylation = gene expression
Hypoacetylation = gene repression
What is the C-value paradox?
The size of the genome is not necessarily related to its complexity.
What are non-coding RNAs? Give some examples.
Any RNA that isn’t translated into a protein (i.e. anything except mRNA)
EXAMPLES: tRNA, rRNA, siRNA
What is mRNA processing?
Splicing out introns before it exits the nucleus and enters the cytoplasm
What bases do introns begin with and end with?
GU —- AG
Describe how small ribonuclear proteins (snRNPs) bind to the mRNA.
U1 binds to the splice donor site, U5 binds to the splice acceptor site, U2, U4 and U6 bind in the middle of the intron
Describe how the intron is removed from the mRNA strand.
The RNA cleaves at the splice donor site and the first G loops round and forms a phosphodiester bond with an adenine residue in the middle of the intron (branchpoint). The mRNA then cleaves at the splice acceptor site and the intron is removed as a lariat structure.
What two structures are added during post-translational modification and why?
7-methylguanylate cap – protects the RNA from degradation – enhances translation
Poly-A-tail – occurs 11-30 bases downstream of an AAUAAA sequence in the mRNA – also acts as protection – added one base at a time
Give an example of a disease that affects one of the structures added during post-translational modification.
Poliomyelitis affects 7-methylguanylate cap recognition during translation
Give an example of a mutation in a splice site which features in human disease.
Beta Thalassemia – splice site mutation in the beta globin gene – caused by imbalances in the relative amounts of alpha and beta chains
Describe how RNA can be used to block mRNA function.
Antisense RNA can be synthesised which binds to the complementary sense RNA forming double stranded RNA thus preventing it from being translated
Describe the formation of siRNA.
You begin with viral dsRNA
DICER cuts the dsRNA into smaller pieces (21-25bp)
AGO proteins then remove the passenger strand only leaving the strand that is anti-sense to the target RNA
The RISC complex associates with the single stranded RNA and cleaves the target mRNA when bound with the siRNA.
What feature of siRNA allows it to be used as a viable therapy?
It is small enough to enter the cell without causing a detrimental cytokine response.
What did the Lin-14/Lin-4 experiment show?
There is genomically encoded miRNA that is involved in gene regulation.
Describe the production of miRNA.
miRNA is made as much bigger RNA known as pri-miRNA
pri-miRNA is processed to form pre-miRNA
Further processing allows the miRNA to be passed into the RISC complex
What is the classic arrangement of miRNA within the genome?
It occurs as a match-bulge-match arrangement in the genome.
One of the matching regions is usually a 3’ UTR and the other is a seed region
Give an example of an alternation in miRNA expression causing disease.
Chronic Lymphoid Leukaemia – deletion of part of gene on chromosome 14 leads to loss of miRNA and promotes CLL
Describe the structure of typical mRNA.
mRNA has a 7-methylguanylate cap at the 5’ end followed by a 5’ untranslated region (UTR)
You then get the coding region in the middle
At the end you get a 3’ UTR and the poly-A tail
What are the stop codons?
UAA, UGA, UAG
What is the Methionine codon?
AUG
What is the significance of Methionine?
It is the first amino acid in virtually all polypeptides
What enzyme is involved in the transfer of an amino acid to tRNA?
Aminoacyl tRNA synthetase
How does this enzyme work?
Aminoacyl tRNA synthetase gets activated (by ATP hydrolysis) to form adenylated amino acid which binds with a molecule of AMP and binds to the relevant amino acid
This adenylated amino acid complex then binds to the tRNA and transfers the amino acid to the 3’ end of the tRNA
The adenylated amino acid and AMP dissociate
What are the three stages of translation?
Initiation – Elongation - Termination
What components are make up the preinitiation complex?
40S ribosomal subunit
Methionine tRNA
eIF2 (initiation factor)
GTP
Describe initiation.
The preinitiation complex recognises initiation factors (eIF4E and G) on the 7MeG and bing to the mRNA
It moves along the mRNA in a 5’ to 3’ direction until it reaches the first in-frame AUG (methionine)
Here the GTP is hydrolysed providing energy to ensure correct base pair matching
The GDP and eIF2 then dissociate causing a conformational change, which allows the binding of the 60S ribosomal subunit to form a full ribosome
Describe elongation.
The next charged tRNA comes and binds to the A site of ribosome
Peptidyl transferase catalyses the formation of a peptide bond between the two amino acids
The tRNA from the P site then dissociates and the ribosome moves along
What proteins facilitate elongation?
The next charged tRNA comes and binds to the A site of ribosome
Peptidyl transferase catalyses the formation of a peptide bond between the two amino acids
The tRNA from the P site then dissociates and the ribosome moves along
What proteins facilitate elongation?
Elongation factors
Describe termination.
When the A site of the ribosome moves over the first in-frame stop codon, a release factor (a type of protein) binds instead of a tRNA
The release factor transfers the growing polypeptide chain to water thus terminating the polypeptide and allowing it to detach from the ribosome
Where is the signal sequence found and what does it consist of?
It is found at the N terminus
It mainly consists of hydrophobic amino acids
Describe how proteins enter the ER.
The signal sequence binds to a signal recognition particle (SRP) and this binding halts translation
The SRP then binds to an SRP receptor on the ER membrane and translation resumes
The binding of SRP to the SRP receptor triggers the assembly of a protein channel through which the polypeptide is threaded into the ER
What extra feature do proteins that are destined to be transmembrane have?
They have an extra signal sequence
What happens once the protein has entered the compartment?
The signal sequence is cleaved
Describe, in full, the post translational modification involved in the production of insulin.
You begin with preproinsulin, which has an N-terminus signal sequence
Once it enters the ER the signal sequence is cleaved and it 3 disulphide bonds form
This makes it proinsulin
Then the C chain is cleaved making it into insulin
You get an A and B chain held together by disulphide bonds
