Nucleic Acids Flashcards
What are the Purines and Pyrimidines?
Purines: Adenine (A), Guanine (G)
Pyrimidines: Cytosine (C), Thymine (T), Uracil (U)
How is a ribose sugar different from a deoxyribose sugar?
The ribose sugar has an -OH on the 2’ carbon; the deoxyribose sugar has a -H on the 2’ carbon.
What is a nucleotide comprised of?
Nitrogenous base + Sugar + Phosphate groups
What does a nucleoside consist of?
base + sugar only
Name the nucleosides
Adenosine Thymidine Uridine Cytidine Guanosine
How are deoxyribonucleotide units joined together?
The 3’ OH of the sugar of one nucleotide is linked to the phosphate group joined by the 5’ OH of an adjacent sugar
In what direction does one talk about a nucleotide chain?
5’ to 3’
What shape does DNA form?
A right-handed double helix
What is the charge distribution of a DNA chain?
The deoxyribose and phosphate groups run along the outside therefore with the negative charges outside
How many hydrogen bonds are shared in the watson-crick base pairs?
A-T = 2 hydrogen bonds G-C = 3 hydrogen bonds
What is a karyotype?
An organised profile of someone’s chromosomes
What is the lowest level of DNA packaging, and what does this consist of?
Nucleosome: DNA wrapped around in histones
What is the name of the process where DNA is copied?
Semi-conservative replication
Outline the process of semi-conservative replication
DNA is unwound by DNA Helicase.
DNA Polymerase enzymes add nucleotides to the 3’ end of a growing chain.
What does DNA polymerase require to generate a new strand of DNA?
A template strand
deoxynucleotide triphosphates (dNTPs)
Primer
How are the energy demands of DNA replication met?
ATP is needed as a source of energy for the DNA Helicase enzyme.
Energy released by the hydrolysis of the triphosphate drives the DNA polymerase enzyme.
What is a nucleoside analogue?
A chain terminator that does not have a hydroxyl group on the 3’ carbon.
Where does DNA replication begin?
At discrete points called origin of replication, forming a replication fork.
Why is the DNA replication fork asymmetric? What are the names of the two strands?
The replication forks produces two template strands. One template strand is being exposed on the 3’ to 5’ direction, allowing the new DNA strand to grow in the 5’ to 3’ direction uninterruptedly. This is called the leading strand.
The other template strand is being exposed in the 5’ to 3’ direction, so the new DNA strand is grows in fragments called okazaki fragments. This is called the lagging strand
Describe the role of primers in DNA synthesis
RNA primers around 10 nucleotides long is synthesised by a DNA polymerase enzyme. The primer is only transient and is removed at a later stage.
For the leading strand, the primer is needed only to start replication at the replication origin.
For the lagging stand, the DNA primes has to start each okazaki fragment. The intermediary RNA primers are erased by a ribonuclease enzyme and is replaced by DNA by repair DNA polymerase. DNA ligase joins new okazaki fragments onto the growing chain.
Describe the error checking mechanism in DNA replication
The DNA polymerase checks the previous base pairing. Any incorrect bases are removed by the 3’ to 5’ exonuclease activity of the DNA polymerase enzyme.
Describe the cell cycle
G1 phase (prior to DNA synthesis) S phase (DNA synthesis) G2 phase (prior to mitosis) M phase (Mitosis)
G0 phase (cells that have stopped dividing, before S phase)
What genes are expressed in all cells?
Housekeeping genes
In gene transcription: what are the names of the DNA strands?
The sense strand is the one that is ‘copied’. The antisense strand acts as a template.
What are the three types of RNA polymerase used for?
RNA Polymerase I: Transcribes rRNA genes
RNA Polymerase II: Transcribes genes encoding proteins into mRNA
RNA Polymerase III: Transcribes tRNA and 5S RNA genes.
How is the level of transcription manipulated? How does this work at a molecular level?
TRANSCRIPTION FACTORS can either be repressors or activators. They bind to the GENE PROMOTER region of the INITIATION COMPLEX. Transcription activators bend the DNA on binding, helping to unwind it.
Transcription factors also allow enzymes to modify chromatin. Inactive chromatin is tightly wound up, active chromatin is loosely wound up.
What is the initiation point for DNA transcription?
TATA
Describe the formation of the Basal Transcription Complex
TFII D recognises TATA, binds to TATA in the minor groove and unwinds it in the direction of transcription only. This is called asymmetric unwinding. TFII D contains TATA Binding Protein and TBP Accessory Factors.
Next, TFII A and B binds tio TFII D bind allowing RNA Polymerase II to bind (with TFII F already bound)
Finally TFII E, H and J bind. TFII H promotes further winding by phosphorylating RNA Polymerase II.
How does histone acetylation correlate with gene expression?
HYPERacetylation correlates with gene expression as it helps loosen the chromatin.
HYPOacetylation correlates with gene repression
What group of transcription factors are important for development?
The Hox family group of transcription factors
What is the initial RNA polymer produced by RNA Polymerase II called?
The Primary transcript or Pre-mRNA or heterogenous nuclear RNA (hnRNA)
Outline primary transcript processing
Introns are spliced out, while exons are joined together. This forms the final RNA. Then a 5’ CAP molecule and poly-A tail is added.
What are the specific base sequences of the splice donor site and splice acceptor site?
Splice donor:
Most exons end in AG. ALL introns start with GU.
AG][GU
Splice acceptor:
Leading to the end of an intron, a run of 15 pyramiding is found, followed by any base, then a C. Finally ending in AG.
Pyr15NCAG][exon
Describe the formation of the spliceosome
The first snRNP, U1, recognises the donor sequence and binds to it.
The next to bind are U2, U4, U5 and U6. U5 binds to the splice acceptor site.
Describe the processes of RNA splicing
The formation of the spliceosome results in the cleavage of the splice donor site (break of phosphodiester bond).
The cleaved end then loops around to an ‘A’ residue in the intron called a branchpoint (phosphodiester bond between 5’ phosphate of the G and the 2’ OH of the A).
The phosphodiester bond between the G at the end of the into and the next exon is then cleaved.
The intron is removed as a lariat structure.
The exons are ligated by an RNA ligase enzyme.
Describe the process of adding a 5’ CAP, with the enzymes for each stage.
1) Hydrolysis of the 5’ triphosphate of mRNA to a diphosphate by the a phosphatase enzyme
2) The diphosphate reacts with the alpha-phosphate of a GTP molecule to form a 5’-5’ phosphate linkage (done by guanyl phosphate)
3) The cap is further modified at the N7 position in the purine ring to form a 7-methylguanylate cap done by the enzyme methyl transferase.
What is the purpose of the 5’ cap?
To protect mRNA and greatly enhance translation as it allows the ribosome to more readily recognise mRNA.
How does the polio virus effect translation?
It transcribes a protease enzyme which its the protein required to bind the 5’ mRNA cap to the ribosome - killing protein translation of indigenous genes. The 5’ end of its own mRNA is structured in a way that it can bind to the ribosomes without the need for a cap.
What is the process of polyadenylation?
The addition of a poly A tail to the pre-mRNA downstream of the polyadenylation sequence (AAUAAA).
Give an example of a disease caused by incorrect splicing. Where is it most common?
What is the physiopathology?
Thalassaemia is an inherited disorder characterised by an imbalance of the relative amounts of alpha and beta global chains, ultimately leading to broken erythrocytes.
It is most common in the mediterranean, S.E Asia and china.
It can cause severe anaemia, extra medullary haumatopoeisis, heptamegaly, hepatospenomagelly and iron overload (haemosiderosis).
What is a RNA molecule that is not translated into a protein called?
Non-coding RNA (ncRNA).
What are the different types of ncRNAs?
- microRNA (controls translation of most genes)
- siRNA/RNAi (viral defence)
- piRNA (involved in epigenetics)
- long ncRNA (important for X chromosome inactivation)
Explain how RNA silencing works
Single-stranded Interference RNA (siRNA/RNAi) anneal to viral RNA causing them to degrade.
1) RNAse III-like endonuclease activity on dsRNA by enzyme Dicer breaks the dsRNA into 21-25 bp fragments
2) ‘Ago’ proteins remove a strand of RNA known as the passenger strand.
3) The remaining targeting strand combines with RISC proteins (RINA-induced silencing complexes)
4) The siRNA (with RISC) bind to the target mRNA. The RISC then cleaves the mRNA, resulting in sliced mRNA.
What are the start and stop codons? What do they code for?
Start is AUG and it codes for methionine (Met). The stop codons are UAA, UAG and UGA, they don’t code for anything.
What direction is mRNA translated in?
5’ to 3’
Describe the structure of tRNAs
tRNAs are smaller RNAs which have a 3’ and 5’ end. It has a ‘clover leaf’ shape held together by hydrogen bonding.
At the anticodon loop there are 3 anti-codons sticking out.
How is a tRNA molecule attached to an amino acid?
An amino acid attaches to an Aminoacyl tRNA synthetase enzyme (E), which uses ATP converting it to AMP (removing two Pi). Forming E-AMP-Amino Acid complex.
This then reacts with a tRNA to form a tRNA-Amino acid complex, leaving AMP and E.
What are the three stages of translation?
Initiation
Elongation
Termination
Explain the process of Translation initiation
1) Dissociation of ribosome subunits into 40s + 60s
2) Assembly of pre-initiation complex. This is aided by enzymes called initiation factors (eIFs). eIF-2 is bound to GTP, which binds to the small rRNA subunit. This forms the pre-initiation complex.
Only met-tRNA can bind to this complex.
eIF-4e and eIF-4g binds to the 5’ cap.
3) eIF-4e and eIF-4g bound to the cap are recognised by the 40s/Met-tRNA/eIF-2 complex
4) The GTP is then hydrolysed. This energy ensures correct base pairing. A conformational change in the complex allows the 60s subunit to bind. GDP and eIF-2 dissociate, leaving the formed ribosome.
What is the site of the ribosome where the initiator is bound called? What is the name of the site adjacent to that?
P site and A (amino acyl) site
Explain the process of Translation elongation
1) Binding of new tRNA to A site on ribosome
2) Catalysis of peptide bond between two amino acids by pepidyl transferase on the 60s subunit.
3) Translocation of pepidyl tRNA to P side (ribosome moves along). This is facilitated by elongation factors (proteins that promote movement using GTP) which also enhance efficiency and accuracy of translation by providing pauses where GTP is hydrolysed, allowing incorrect base pairs to dissociate.
How is translation checked?
1) GTP hydrolysis from eIF-2 ensuring Met pairing correct
2) GTP hydrolysis by elongation factors provides pauses allowing incorrect base pairs to dissociate.
Explain the process of Translation termination
1) Recognition of stop codon by release factors binding to empty A site coding for stop.
2) Release of peptide chain as pepidyl transferase catalyses the transfer of the completes protein to water and releases it from the ribosome
3) Dissociation of release factors and ribosomes
What is a polyribosome?
Ribsomes working together on a mRNA molecule
What antibiotics inhibit Protein translation?
Steptomycin inhibits initiation
Tetracycline inhibits amino acid-tRNA binding
How are transmembrane protein mRNA different?
The first 20-24 amino acids are the ‘signal sequence’ of hydrophobic amino acids
How are secretory and transmembrane proteins synthesised?
1) Recognition of signal sequence by a protein-RNA complex called the signal-recognition particle (SRP) pauses translation
2) SRP binds to a receptor at the RER surface, translation resumes
3) Translocation of the growing peptide into the lumen of the RER as binding of SRP triggers formation of a protein channel.
4) Cleavage of the signal sequence by signal peptidase enzyme
How does the primary structure of a transmembrane protein differ from that of a secretory protein?
The transmembrane protein will have more than one one hydrophobic sequence, anchoring it in the vesicle.
What are the ways proteins can be modified post-translation?
- Disulfide bond formation
- Glycosylation
- Phosphorylation
- Addition of lipid groups
- Hydroxylation
What is DNA cloning?
A method of selectively amplifying DNA sequences to generate homogenous DNA populations.
What are the two ways of accomplishing DNA cloning?
Cell-based (in vivo) and Cell-free (in vitro)
What are the steps involved in cell-based DNA cloning?
1) Construction of recombinant DNA:
- cut target DNA and replicon with same specific restriction endonuclease enzymes
- this creates complementary sticky ends
- mix DNA fragments together and join them using DNA ligase
2) Transformation of the recombinant DNA into host cells (bacteria or yeast).
- heat shock therapy
3) Selective antibiotic resistance marker in the replicon means that when bacteria are placed in agar treated with that antibiotic, only those with the replicon survive.
4) Expansion of the cell culture and isolation of recombinant DNA
How do restriction endonuclease enzymes work?
Type II restriction endonuclease enzymes cleave DNA at specific recognition sequences, which are usually 4-8 bp palindromic sequences. These produce blunt and sticky ends.
Why have restriction endonucleases evolved?
They are one half of bacterial restriction-modification systems. Host DNA is protectedly methylation of the bases in the restriction endonuclease site by a specific methylase. Restriction endonuclease enzymes only cleave unmethylated DNA.
Explain how can DNA molecules be separated?
By a process called electrophoresis:
DNA is placed in a solution, and moves towards the anode (+ve) as it has a negative backbone. This allows DNA to be separated by its size as smaller fragments are retarded less and so travel aster.
The solution used is a porous gel matrix (agarose/polyacrylamide gel)
What are hybridisation assays?
Nucleic acid hybridisation is a method for detecting specific sequences in which homologous single-stranded DNA or RNA molecules hybridise to form a double-stranded molecule.
What is a standard assay?
Standard assays involves a labelled nucleic acid probe to identify homologous target molecules in a mixture of unlabelled nucleic acids.
What are the different hybridisation assays?
- Southern blotting (DNA target, DNA probe)
- Northern blotting (RNA target, DNA probe)
- Colony blotting (bacterial DNA target, DNA probe)
- Tissue in-situ hybridisation (RNA target, RNA probe)
- Chromosome in-situ hybridisation (chromosome target, DNA probe)
- Reverse hybridisation (immobilised DNA or oligonucleotide probe, target DNA solution)
What is restriction fragment length polymorphism
A technique where different restriction locations on different alleles produce different length DNA fragments. These can be analysed to give the genotype of an individual.
What is hybridisation stringency? Would you want high or low stringency?
The degree to which the nucleotides must match between the probe and target sequence before they base-pair.
Therefore you would want high stringency for optimising accuracy.
How is hybridisation stringency manipulated? What factors does this depend upon?
It is manipulated by changing the temperature. Stringency is improved by increasing the temperature, but this must be balanced with the denaturation of the DNA probe. The factors this depends upon are:
- strand length (longer = more h-bonds to break)
- base composition (C-G pair more h-bonds than A-T)
- chemical environment (monovalent cations such as Na+ neutralise charge on phosphate backbone, stabilising DNA duplex)
What is Tm (in the context of hybridisation stringency)? And what is this value for human DNA? At what temperature is hybridisation carried out?
Tm is the temperature at which 50% of the DNA is double stranded and 50% is single. This is 87 degrees C for mammalian DNA. Hybridisation is carried out <25 degrees C below Tm
What are the steps involved in Cell-free DNA cloning?
PCR:
1) 2 primers are needed (complementary to each strand)
2) Primers are annealed to heat denatured DNA (94 degrees C) by lowering temperatures to 50-60 degrees C
3) Thermostable Thermophilus aquaticus DNA polymerase + dNTPs extent 5’ to 3’ from the primers to generate new strands at 72 degrees C.
4) Raise temperates to 94 degrees C to denature and repeat process
What are the criteria for PCR primer selection?
- Length (usually around 20 nucleotides)
- Base composition (avoid tandem repeats that can form hairpins, and similar GC% for Tm)
- Avoid complementary bases at 3’ ends as primers would bind to each other
What are the applications for PCR?
- Restriction Fragment Length Polymorphisms
- Detecting point mutations
- cDNA cloning
- Gene expression (reverse transcription PCR)
- DNA sequencing
- DNA microarrays
What is a DNA microarray?
A collection of microscopic spots containing oligonucleotides. By treating the microarray with fluorescent DNA, we can see what genes the DNA makes. It is used for expression profiling by taking mRNA and then converting to CDNA. It can also look for single nucleotide polymorphisms (SNPs) and see what genes are expressed in cancer cells)
Describe the process of adding a 5’ CAP, with the enzymes for each stage.
1) Hydrolysis of the 5’ triphosphate of mRNA to a diphosphate by the a phosphatase enzyme
2) The diphosphate reacts with the alpha-phosphate of a GTP molecule to form a 5’-5’ phosphate linkage (done by guanyl transferase)
3) The cap is further modified at the N7 position in the purine ring to form a 7-methylguanylate cap done by the enzyme methyl transferase.
What proteins does splicing depend upon?
Ribonuclear proteins - proteins which have small ribosomes embedded in them. (snRNP)
What proteins does splicing depend upon?
Ribonuclear proteins - proteins which have small ribosomes embedded in them. (snRNP)
How many genes are there in the human genome?
23,000
What end of the tRNA are amino acids bound to?
3’ end
How many base pairs does DNA have per helical turn?
10
What is the width of the DNA double helix?
2nm
What is the lowest level of DNA packaging, and what does this consist of?
Nucleosome: DNA wrapped around in histones in a left-handed superhelix
How are histones attached to DNA?
Histones are positively charged; they interact with the negative backbone of DNA.
The CORE region of DNA winds round the histone OCTAMER, the LINKER regions link together these complexes.
What is the width of the DNA double helix?
2nm
Which structures give rise to a triadic junction?
T-tubule and two adjacent sarcoplasmic reticulum
