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Nucleic Acids Flashcards

1
Q

Explain how RNAi is developed and works

A
  1. siRNA or microRNA enter the cytoplasm
  2. Break down into fragments by DICER and endonuclease
  3. Creates 3’ overhangs
  4. Ago proteins attach to the overhangs
  5. Formation of RNA induced silencing complexes (RISC) with ATP
  6. Attachment to complementary RNA
  7. cleavage and silencing of the RNA
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2
Q

what is the difference between siRNA and microRNA

A

siRNA is made exogenously and cuts mRNA at 1 point

microRNA is made endogenously and cuts at multiple points + hairpin structure

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3
Q

Describe microRNA

A

genetically encoded siRNA which was first seen in C. Elegans (Lin4)
3’ overhang by 2 nucleotides

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4
Q

Explain how microRNA works

A
  1. miRNA is transcribed by RNA polymerase II into a pre-mRNA
  2. Processing by RNase III endonuclease (Dresha) complex with DIGeorge syndrome critical region
  3. Transport to the cytoplasm
  4. RISC formation
  5. Binding via the seed region (rest is not complementary)
  6. Permanent binding
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5
Q

Give an example of how miRNA is involved in human disease

A

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

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6
Q

Why is analysis of DNA important

A

Used for personalised mediciein e.g. and increase in HER2 can lead to breast cancer
Herceptin can be used for those with HER2

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7
Q

What are restriction endonuclease

A

Enzymes that cleave DNA at specific sequences
Only cuts unmethylated DNA
Produces blunt or sticky ends

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8
Q

What is electrophoresis

A

Technique to separate DNA fragments
-ve DNA is attracted to a +ve charge at the anode
Smaller fragments will travel faster and further

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9
Q

Describe the process of in vivo cloning

A
  1. cut the target DNA using REs
  2. Cut the replicon (replicates out of the host e.g. plasmid bacteriophage) with the same RE
  3. combine fragments using DNA ligase
  4. transformation of the recombinant DNA
  5. Selective propagation of the colonies
  6. expansion via culturing
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10
Q

Explain one technique for selective propagation in in vivo cloning

A

Antibiotic resistance in the recombinant gene so exposure to the antibiotic will kill those who did not take up the gene

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11
Q

Describe the process for in vitro cloning

A

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

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12
Q

What are the 3 things required for PCR

A

Oligonucleotide primer
dNTP
Taq polymerase

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13
Q

What can DNA cloning be used for

A 
sequence DNA
Detect point mutation
DNA microarrays
cDNA cloning
Typing genetic markers
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14
Q

What are microarrays

A

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

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15
Q

How does hybridisation work

A
  1. denature the nucleic acid
  2. single-strand reacts with nylon or nitrocellulose to immobilise
  3. complementary nucleic acid (probe)
  4. H-bonds form between the target and probe
  5. electrophoresis
  6. probes show radioactivity or fluorescentivity
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16
Q

What are the different types of hybridisation

A 
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
17
Q

What is the entry point for a ribosome during translation

A

7MetG cap

18
Q

What 3 materials are required for translation

A

mature mRNA, charged tRNA, ribosome

19
Q

What are the start and stop codons

A 
Start = AUG = Met
Stop = UAA, UAG, UGA
20
Q

Describe the structure of tRNA

A

Clover shaped
Carries and amino acid (Charged)
anticodon (anti-parallel)
64 RNAs

21
Q

Explain how tRNA becomes charged

A
  1. Amino acid binds to aminoacyl tRNA synthase
  2. Aminoacyl tRNA synthase cleaves pyrophosphate from ATP and binds remaining AMP to AA
  3. Amino acids is adenylated
  4. Adenylated AA is attached to tRNA
  5. Aminoacyl tRNA synthase and AMP then detaches leaving the charged tRNA
22
Q

What are the 3 stages of translation

A

Initiation
Elongation
Termination

23
Q

Explain the process of initiation in translation

A
  1. ribosomal unit dissociates into 40S and 60S units
  2. initiation factor 4G and 4E bind to the cap
  3. Charged tRNA, eIF2, GTP, 40S recognise the structure
  4. Reads until AUG to form the pre-initation complex
  5. GTP is converted to GDP which then binds to 60S
  6. met-tRNA binds to the P-site on ribosomes
24
Q

Explain the process of elongation in translation

A
  1. The next tRNA binds to the A site
  2. peptide transferase creates a peptide bond
  3. elongation factors move along the strand using GTP (translocation)
25
Q

What is the function of GTP hydrolysis in elongation during translation

A

gives time for dissociation of incorrect amino acids

26
Q

What doe ribosomes split into during translation in prokaryotes

A

30S and 50S

27
Q

Explain the process of termination in translation

A
  1. Stop codon attracts release factors (no tRNA for stop codon)
  2. binding to the A site of the ribosome
  3. peptide transferase binds water to the final amino acid (carboxyl group)
  4. Translocation complex dissociates
28
Q

What molecules are involved in modification after translation

A

signal sequence or signal recognition proteins (20-24AA)

29
Q

Explain the process of modification after translation

A
  1. SRP is detected by the SRP receptor in the RER to stop translocation
  2. SRP binds to the receptor
  3. Translocation resumes
  4. Polypeptide moves to the lumen of the RER
  5. SRP degraded
  6. Polypeptide folded and cleaved
  7. Further modification or transmembrane (further hydrophobic groups added)§
30
Q

Describe insulin modification

A

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

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