Nucleic Acids Flashcards
Compare deoxyribose to ribose
Deoxyribose lacks an oxygen atom that ribose has
What is the base and phosphate attached to on the sugar
base - 1’ C
phosphate - 5’ C
Give the purines and pyrimidines
purine (big) - A and G
pyrimidine (small) - C and T
What are the functional groups present on the bases
A - NH2 G - =O, NH2 C - NH2 T - =O, CH3 U - O
What are nucleosides
Sugar and base with no phosphate
Give the corresponding nucleosides for each base
A - adenosine G - guanosine C - cytidine T - thymidine U - uridine
How are nucleotides linked to one another
3’ OH of one is linked to the 5’ OH phosphate of another
Describe the structure of DNA
Asymmetry due to 5' and 3' right handed double helix anti-parallel chains bases point inwards and perpendicular (-ve inside) 10 bp per helical turn minor and major grooves
How many H-bonds are there between bases
A-T = 2 C-G = 3
How is DNA analysed
Separation by heating or low salt and then re annealing/hybridised by cooling or high salt
karyotype can be found
How is DNA arranged in the nucleus
Tightly packaged with histones (+ve) to form chromatin. 8 core proteins.
- DNA
- nucleosome
- 30nm fibre
- chromatin
- chromosome
Explain what semi-conservative replication is
Each daughter DNA inherits one old and one new strand
What is the function of DNA helicase
unwinding of the DNA helix using ATP
In which direction is DNA replicated
5’-3’ as nucleotides are added to the 3’ end by DNA polymerase
What does DNA polymerase require
Template strand
Oligonucleotide primer
Deoxynucleotide triphosphates (dNTPs)
Describe the replication fork
Begins at the origin of replication
Asymmetric replication as both strands are synthesised 5’-3’
Explain the process in the replication fork
Leading strand is synthesised continuously while the lagging strand is synthesised as Okazaki fragments
- DNA primase synthesises a short RNA primer fragment
- Ribonuclease removes the previous primer 5-3- exonuclease activity
- repair DNA polymerase replaces the primer with DNA
- DNA ligase joins the two fragments using ATP
Explain the purpose of the following: single strand DNA binding protein, sliding clamp, exonuclease
SSDBP - prevents the single strands from folding
Sliding clamp - prevents DNA polymerase from shifting or falling
3’-5’ exonuclease - checks and fixes mutations
Describe the replication of E. Coli
Begins at OriC
2 replication forks in opposite directions
B-directional
Describe the replication of eukaryotes
multiple replication origins distributed at intervals
each give bi-directional forks
Describe the cell cycle
G1 - 10 hrs of gap phase (G0 - cells stop dividing) S - 9 hrs of DNA synthesis G2 - 4 hrs of gap phase M - 1 hr mitosis
Describe the stages of mitosis
G2 - chromosomes not visible
Early prophase - chromosomes pair + crossing over
Late prophase - chromosomes condense and become visible, spindle fibres move
Metaphase - chromosomes aligned on the central plane
Anaphase - sister chromatids pulled apart
Telophase - sister chromatids move to opposite poles
Cytokinesis
Name 4 nucleoside analogues and explain how they work
AZT - N3 group for HIV Acyclovir - no ring sugar for herpes ddC - H group for HIV Cytosine Arabinose - tetrose sugar for chemotherapy Absence of a 3' OH group
Explain the basic differences between DNA and RNA
DNA - deoxyribose, RNA - ribose
AGCT, AGCU
long and double stranded, short and single stranded
RNA: rRNA, tRNA, mRNA
Explain what transcription means
Production of mRNA from DNA
Growth from 5’-3’ (Sense)
List the major functional classes of RNA and the classes of RNA polymerases involved in synthesising each of these.
I - rRNA
II - mRNA
III - tRNA and 5S RNA genes
Grooves in the helix allows for adherence to the strands
Explain what a gene promoter is
DNA sequences at which the initiation complex assembles at. Gives the initiation point. e.g. TATA
Explain what is meant by a transcription factor
Proteins that regulate genes acting collectively to bring about expression. They activate or repress transcription or may model chromatin. May be signalled by hormones, growth factors, stress etc.
What is the process involved in transcribing a eukaryotic gene
- TF II D unwinds the DNA to widen the minor groove
- TF II A and TF II B bind.
- RNA polymerase binds to TF II B
- Other TFs bind to promote further unwinding. TF II H further unwinds the DNA helix (E,F,H,J)
Explain how acetylation can affect transcription
Hyperacetylation - expression
Hypoacetylation - repression
What does TF II D contain and how does it assist transcription
TBP (TATA binding protein)
TAF (TBP accessory factor)
unwinds the DNA asymmetrically so that transcription is unidirectional
How does aspirin work
inhibits the breakdown of IkB so NFkB remains in the cytoplasm so cannot initiate transcription of cytokine genes
What is the structure of pre-mRNA
GU at the start
A in the middle
15 pyrimidines, random nucleotide, CAG at the end
What does RNA processing use
ribonucleic proteins snRNPs
Explain the events that take place in pre-mRNA processing
- U1 binds to the splice donor sequence (GU)
- U2, U4, U5, U6 binds to form the spliceosome (U5 binds to CAG)
- Cleavage of the splice donor sequence and formation of a branch point (phosphodiester bond) at A between 5’ P on G and 2’Oh on A
- PD bond between intron and exon at the end is cleaved to release lariat intermediate
- DNA ligase joins the exons together
What is the purpose of the CAP and PolyA tail
CAP protects the mRNA and enhances translation
PolyAAA = added 11-30 bases from AAUAAA
What are the splice donor and acceptor sites
splice donor = GU where U1 binds
spice acceptor = CAG where U5 binds
Explain how mutations in splice sites feature in polio
Polio interferes with the recognition of CAP and invades the nervous system to cause total paralysis
What proportion of mutations are in splice donor sequences
1/3
Explain how mutations in splice sites feature in thalassaemia
Inherited disorder where there is an imbalance in globin chains
Explain how mutations in splice sites feature in DMD
Duchenne muscular dystrophy
Dystrophin gene is mutated (most deletion of axons) causing prematurely aborted dystrophin protein synthesis
How is DMD treated
Alternative splicing sp that exons are purposefully skipped but the dystrophin remains functional
Does genome size = complexity
No
What is ENCODE
Encyclopaedia of DNA elements
Identifies all regions of transcription factor association, chromatin structure and histone modification
What proportion of introns are functional
20% of introns = functional
60% = unknown function
What is ncRNA
non-translated RNA that is highly abundant and important
e.g. tRNA, rRNA, microRNA, sn, pi, long ncRNA
Explain X-inactivation
1/2 X chromosomes in females are inactivated (packaged so one is transciptionally inactive) - heterochromatin
choice of which X chromosome is random
Controlled by long ncRNA
What is long ncRNA
ncRNA >200 n
used in C inactivation/lyonisation
What is the experimental support for small ncRNA
Nematode Caenorhabditis elegans is a free living nematode(worm) that is one of the simplest organisms with a nervous system
Used to map the connectome and fate map
dsRNA is a silencing trigger to C. elegans
What is antisense RNA
Transcribing the reverse of a gene
It forms H bonds with the sense RNA to prevent translation
Forms dsRNA
Used in plants against viruses
What is RNAi
A small single stranded RNA that anneals to viral RNA to cause degradation (microRNA + siRNA operate via RNAi)
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 hybridisation stringency
precision to which the hybridisation procedure can distinguish between similar nucleic acid sequences
How can hybridisation stringency be increased
Increase in temperature and a low salt concentration
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
