Nucleic Acids Flashcards
Explain how RNAi is developed and works
- siRNA or microRNA enter the cytoplasm
- Break down into fragments by DICER and endonuclease
- Creates 3’ overhangs
- Ago proteins attach to the overhangs
- Formation of RNA induced silencing complexes (RISC) with ATP
- Attachment to complementary RNA
- cleavage and silencing of the RNA
what is the difference between siRNA and microRNA
siRNA is made exogenously and cuts mRNA at 1 point
microRNA is made endogenously and cuts at multiple points + hairpin structure
Describe microRNA
genetically encoded siRNA which was first seen in C. Elegans (Lin4)
3’ overhang by 2 nucleotides
Explain how microRNA works
- miRNA is transcribed by RNA polymerase II into a pre-mRNA
- Processing by RNase III endonuclease (Dresha) complex with DIGeorge syndrome critical region
- Transport to the cytoplasm
- RISC formation
- Binding via the seed region (rest is not complementary)
- Permanent binding
Give an example of how miRNA is involved in human disease
E.g. chronic lymphoid leukaemia (CLL)
Deletion of part of a gene on chromosome 14 leads to loss of miRNA. Supplementing the miRNA to nude mice that have CLL, you can get rid of the cancer
Why is analysis of DNA important
Used for personalised mediciein e.g. and increase in HER2 can lead to breast cancer
Herceptin can be used for those with HER2
What are restriction endonuclease
Enzymes that cleave DNA at specific sequences
Only cuts unmethylated DNA
Produces blunt or sticky ends
What is electrophoresis
Technique to separate DNA fragments
-ve DNA is attracted to a +ve charge at the anode
Smaller fragments will travel faster and further
Describe the process of in vivo cloning
- cut the target DNA using REs
- Cut the replicon (replicates out of the host e.g. plasmid bacteriophage) with the same RE
- combine fragments using DNA ligase
- transformation of the recombinant DNA
- Selective propagation of the colonies
- expansion via culturing
Explain one technique for selective propagation in in vivo cloning
Antibiotic resistance in the recombinant gene so exposure to the antibiotic will kill those who did not take up the gene
Describe the process for in vitro cloning
Uses polymerase chain reaction (PCR)
Primer must be 20N and tandem repeats should be avoided
A non-complementary end to 3’ so it binds to 5’
1. denaturation at 94 to break H bonds
2. annealing at 50-60 to allow annealing between strands and dNTPs
3. Elongation at 72 for tax polymerase to form PD bonds
What are the 3 things required for PCR
Oligonucleotide primer
dNTP
Taq polymerase
What can DNA cloning be used for
sequence DNA Detect point mutation DNA microarrays cDNA cloning Typing genetic markers
What are microarrays
Monitors expression levels for genes
Collection of dots that represent single genes sprayed on a surface e.g. a glass slide
Expression profiling
SNP detection array - SNP in genomes of the population
How does hybridisation work
- denature the nucleic acid
- single-strand reacts with nylon or nitrocellulose to immobilise
- complementary nucleic acid (probe)
- H-bonds form between the target and probe
- electrophoresis
- probes show radioactivity or fluorescentivity
What are the different types of hybridisation
Southern blot - DNA, RNA Northern blot - RNA, DNA Coloney blot - Bacterial DNA, DNA Tissue in situ - RNA x 2 Chromosome in situ - chromosome, DNA reverse - DNA x 2
What is the entry point for a ribosome during translation
7MetG cap
What 3 materials are required for translation
mature mRNA, charged tRNA, ribosome
What are the start and stop codons
Start = AUG = Met Stop = UAA, UAG, UGA
Describe the structure of tRNA
Clover shaped
Carries and amino acid (Charged)
anticodon (anti-parallel)
64 RNAs
Explain how tRNA becomes charged
- Amino acid binds to aminoacyl tRNA synthase
- Aminoacyl tRNA synthase cleaves pyrophosphate from ATP and binds remaining AMP to AA
- Amino acids is adenylated
- Adenylated AA is attached to tRNA
- Aminoacyl tRNA synthase and AMP then detaches leaving the charged tRNA
What are the 3 stages of translation
Initiation
Elongation
Termination
Explain the process of initiation in translation
- ribosomal unit dissociates into 40S and 60S units
- initiation factor 4G and 4E bind to the cap
- Charged tRNA, eIF2, GTP, 40S recognise the structure
- Reads until AUG to form the pre-initation complex
- GTP is converted to GDP which then binds to 60S
- met-tRNA binds to the P-site on ribosomes
Explain the process of elongation in translation
- The next tRNA binds to the A site
- peptide transferase creates a peptide bond
- elongation factors move along the strand using GTP (translocation)
What is the function of GTP hydrolysis in elongation during translation
gives time for dissociation of incorrect amino acids
What doe ribosomes split into during translation in prokaryotes
30S and 50S
Explain the process of termination in translation
- Stop codon attracts release factors (no tRNA for stop codon)
- binding to the A site of the ribosome
- peptide transferase binds water to the final amino acid (carboxyl group)
- Translocation complex dissociates
What molecules are involved in modification after translation
signal sequence or signal recognition proteins (20-24AA)
Explain the process of modification after translation
- SRP is detected by the SRP receptor in the RER to stop translocation
- SRP binds to the receptor
- Translocation resumes
- Polypeptide moves to the lumen of the RER
- SRP degraded
- Polypeptide folded and cleaved
- Further modification or transmembrane (further hydrophobic groups added)§
Describe insulin modification
undergoes disulphide bond formation in the ER and golgi body
Proteolytic cleavage in secretory vesicle
Chain C is released into the cytoplasm and then the blood so it can be used to detect insulin levels
