DNA structure & function Flashcards
(24 cards)
- Explain how the structure of DNA allows its role as the key information storage molecule
- Transcription
- DNA replication
- DNA repair
- DNA recombination
- Gene regulation - regulated gene activity, which gene is active and when
- Describe the structure of DNA (list and be able to recognise the three components), describe its directionality and secondary structure (the overall structure of a molecule of DNA)
> DNA structure
- Phosphate group
- deoxyribose sugar
- base
Structure
- double-stranded and antiparrallel
- phosphate attached to 5’ carbon and other phosphate at 3’ carbon
- purine pair with pyrimidines and keep width of DNA similar
- AT = 2 H bonds
- GC = 3 H bonds
- AT bond is slightly weaker because of only 2 H bonds
- Each strands are linked by covalent phosphodiester bonds (sugar OH and phosphate) which are stronger than the H bonds
- Recognise which base is a pyrimidine and which a purine. List the bases used in DNA and RNA and how they pair
> Nitrogenous bases
- Adenine & Guanine = Purines (2 rings)
- Cytosine & Thymine & Uracil = Pyrimidines (1 ring)
> Nuceotide role
- components of RNA & DNA
- cofactors of metabolism (carry electrons - NAD+ and FAD+)
- energy storage and use - unit of energy (ATP)
- Explain how the structure of DNA allows its role as the key information storage molecule
- Describe the key aspect of the structure that allows it to direct its own replication
- Describe the structure of RNA (list and be able to recognise the three components), describe its directionality and secondary structure
> Structure of RNA
- Ribose pentose sugar
- Nitrogenous base (purine or pyrimidine)
- Phosphate group
> Secondary Structure
- RNA often forms hairpins and loops and is single-stranded
- They,can have,modified bases,as, well
- They can,bind with,proteins to,form, large,complex structures, e.g., Ribosomes,
Ribosomes, predominantly,made of,RNAs and,some, proteins;,riboprotein, macromolecules.,,
> Directionality of RNA
- 5’ end is a phosphate group
- 3’ end is an OH group coming off 3rd carbon on sugar molecule
- Compare and contrast the structure of RNA with DNA (the components and the overall secondary structure)
- List several different roles of RNA in the cell
- Part of splicing machinery
- Gene expression - miRNAs can bind to mRNA and regulate its expression
- Translation
- Transcription
- Genetic material for viruses
- DNA replication
- Explain what it means that DNA replication is it semi-conservative
Semiconservative DNA replication means that when one DNA molecule replicates, it produces two identical daughter molecules, each containing one original strand and one newly synthesized strand.
Only need one strand of DNA to know what other strand is
Only nucleic acids can do this
Each strand is a template for other strand
- Describe the roles of the DNA replication proteins β-sliding clamp, clamp loader, DNA polymerase I, DNA polymerase III
- Describe the differences in structure and function of DNA polymerase I and III
> DNA polymerase I functions in replication, repair and recombination
- single protein/has one subunit thats wraps around DNA like a hand with the active site in the centre (palm)
- faster and has higher processitivity –> runs along DNA longer
- Has extra domain allowing 5’—>3’ (on template strand) exonuclease activity, so can either add (polymerase activity) or remove base
- mainly used for repair, not much in replication
> DNA polymerase III is main one in replication - synthesises leading and lagging strands
- it is a complex of other subunits (so has higher molecule weight) –> DNA replication machinery
- Yellow subunits - pol III core
- Purple subunits - beta-sliding clamp
- Pol III core is attached to a beta-sliding clamp
- Pol III core & beta-sliding clamp are attached to the clamp loader
- clamp loader is composed of 2 subunits that help enzymes run across DNA for a long time
> EXONUCLEASE ACTIVITY DIFFERENCES
- Refers to removal of base
- DNA POL I has 5’ –> 3’ exonuclease activity
- Both DNA POL I & DNA POL III has 3’—>5’ (proofreading) exonuclease activity
- Explain how a DNA polymerase catalyses the formation of the DNA backbone
- DNA extends 5’ –> 3’ direction
- All DNA and RNA polymerases have 2 Asp residues that interact 2Mg2+ ions which coordinate with oxygen of phosphate group of incoming nucleotide
- frist Mg2+ ion coordinates with oxygen on sugar of growing DNA strand and the oxygen on alpha-phosphate of the incoming nuceotide as the sugar oxygen attacks the alpha phosphate molecule
- the second Mg2+ ion coordinatesd with last 2 phosphates of nucleotide that break off to form pyrophosphate
- bond between alpha phosphate and oxygen of sugar becomes a phosphodiester bond
The DNA polymerase can only extend at 3’ end of sugar so requires a short RNA primer to get process started (DNA polymerase still moves in 5’–>3’ direction tho)
Reaction is processive meaning it will continuosly synthesise phosphodiester bonds along the strand
- Describe the roles of the DNA replication proteins DNA polymerase I, DNA polymerase III, DnaA, DNA helicase, SSB, DNA topoisomerase (gyrase), primase, DNA ligase.
- Describe the initiation of DNA replication at the origin of replication
- For replication to occur, DNA needs to be seperated first - this occurs at specific sites of DNA called origins of replication
- Origin of replication opens up to form replication bubble with a replication fork at each end that will open up DNA as it moves along and eventually joins the other replication forks (like a zipper)
> Origin of replication sequence in E.coli is called OriC
- it has two sections - DUE (orange) region and a grey section
- DNAa protein binds to grey section longitudinally that is rich in A and T and forms a helix to twist DNA and create TENSION
- TENSION causes DUE region of DNA to seperate which is rich in AT base pairs
- One DNAb helicase each then bind to each replication fork at each end (helicase is a spinning motor of 6 subunits hydrlysing ATP as it unwinds DNA)
(E.coli only has 1 replication bubble unlike eukaryotes that have many because of larger chromosomes)
Ahead of helicase is DNA gyrase/Topoisomerase II which relieves tension as the replication fork continues to unwind so process can continue
Behind helicase is SSB proteins attaching to single strand to prevent nuclease attack
RNA primase then places a short primer so DNA polymerase III can start replication from 5’ to 3’ end as a new strand (because DNA strands are antiparallel)
- Explain the difference between the replication of the two strands
THe DNA polymerase III ALWAYS replicates from 5’—>3’ end for NEW STRAND so reads template strand from 3’—>5’
- but this means one strand will be replicated against the replication fork ( half the fork hasnt even opend yet), so it will go backwards essentially (instead of reading template from 3’—>5’, it still does that but starts closer to the 5’ end) and synthesise 5’—>3’ segments of the new strand leaving gaps in between wherever the RNA primer is present (when this primer is removed the gap can be filled) - this is the lagging strand
- RNA primer needs to continously place new primers so this can work
- The short fragments of DNA synthesised by DNA pol III are called okazaki fragments
In the other strand (leading strand) DNA pol III travels continuosly in the same way as the replication fork
> DNA pol 1 removes RNA primer in 5’—>3’ direction (RNAase enzyme can also do this)
DNA ligase will create final phosphodiester bond to complete backbone
- Describe the three-dimensional organisation of the replication fork
All enzymes must travel in the direction of the replication fork.
Even though the two DNA strands are antiparallel, the enzymes are organized so they all move together as the replication fork progresses.
The β (beta) clamp assists DNA polymerase in maintaining processivity, allowing it to replicate long stretches of DNA without dissociating.
All enzymes involved in replication are coordinated through the clamp loader complex, which helps assemble and recycle components like β clamps and DNA polymerase at the correct locations.
To allow both DNA polymerases (for leading and lagging strands) to move together with the helicase, the lagging strand template loops around. This loop ensures that both polymerases are physically moving in the same direction, even though the lagging strand is being synthesized discontinuously.
Once the lagging strand polymerase reaches the RNA primer of the previous Okazaki fragment, it releases the DNA, and the loop collapses. At this point, other enzymes — DNA Pol I, primase, and ligase — coordinated by the clamp loader, finish processing:
DNA Pol I removes the RNA primer and replaces it with DNA.
DNA ligase seals the nick between fragments.
Meanwhile, primase places a new RNA primer further downstream (closer to the replication fork).
The clamp loader places a new β clamp at the site of the new RNA primer. A new lagging strand loop forms, and DNA Pol III reattaches to begin synthesis of the next Okazaki fragment.
- Define telomere and telomerase, and briefly explain what problem they are solving
Structure of a chromosome:
- Centromere linking the two chromatids (each chromatid is equivalent to one DNA molecule which after replication is bound to a sister chromatid by a centromere and together the complex is called a chromosome)
- Telomeres at the end of the chromatids to protect the ends
> When replication fork moves to end of the chromosome lagging strand isnt actually replicated untill the end
- when the last RNA primer of okazaki fragment is removed it cant be replaced with DNA because theres no 3’ end available for DNA polymerase —> subsequent rounds of replication for the new daughter DNA molecules will yield shorter and shorter chromosome until there’s no functional chromosome
–> so telomerase addes new DNA at the ends called telomeres
- it synthesises a very unusual DNA structure at the ends of eukaryotic chromosomes
- has a lot of repetitive DNA elements and a loop structure looping back on itself
- The 3’ end extends down into the bubble that opened up and base pairs with the adjacent strand at the bubble –> stabilised by those proteins bound over the top
- Explain the mechanisms for how the fidelity of DNA replication is maintained, and why this is important
> Replication of DNA must be extremely accurate, DNA is replicated so all body cells have the same genome, if DNA had many errors, all our cells would have a slightly different genome
> DNA Polymerases are very accurate thru two key mechanisms
1. Binding of nucleotides
- what determines which nucleotides bind the active pocket of DNA polymerase III?
- it is a combination of correct shape and charge, especially the H bonds that forms with the exisiting nucleotide
- Proofreading (3’—>5’ exonuclease activity)
- It isnt really “proofreading” because DNA pol doesnt scan along for errors
- when a wrong base is paired it doesnt fit well into the active site of the DNA pol so the polymerase slides back and shifts the wrong base into its exonuclease active site so it can remove it and reposition itself again by sliding forward to normal and wait for the correct nucleotide to incorporate
> DNA pol I and III both have this exonuclease ability but DNA pol I can do 5’—>3’ exonuclease activity so it can move forward whilst replacing incorrect bases while traveling
SUMMARY:
🧬 DNA Polymerase I vs III – Exonuclease Direction
🧪 DNA Polymerase III
Has 3′ → 5′ exonuclease activity only.
➤ Moves “backward” (opposite the direction of synthesis) to remove mismatched bases just added.
Used for proofreading during replication.
🔁 Think: “Backspace once when you make a typo.”
🛠 DNA Polymerase I
Has both:
3′ → 5′ exonuclease activity → for proofreading (same as Pol III).
5′ → 3′ exonuclease activity → unique to Pol I.
This 5′ → 3′ exonuclease allows DNA Pol I to:
Remove RNA primers from the 5′ end of Okazaki fragments.
Remove damaged DNA ahead of synthesis.
And then replace it with new DNA moving forward (5′ → 3′ synthesis).
- Compare the roles and functions of the DNA replication proteins DNA polymerase I and DNA polymerase III
- Describe the important features of an E coli plasmid vector for use in cloning DNA
MOLECULAR CLONING
> is used to assemble recombinant DNA molecules and amplify them by replication within host cells
1. Scientists identify specific gene of interest they wish to clone (can be a gene of interest, regulatory element (e.g. promoter region))
2. The vector is the piece of DNA into which the gene of interest will be inserted. This is typically a circular dsDNA molecule known as a plasmid, which occurs naturally in bacteria.
> COMMON PLASMID ELEMENTS:
- Antibiotic resistance gene
- origin of replication enabling replication of plasmid inside bacterial cell
- promoter region to drive gene expression of the gene that will be inserted downstream of it
- Restriction sites - specific DNA sites where DNA can be cleaved later - these sites lie downstream of promoter region
- Describe the overall process for cloning a piece of DNA into a plasmid vector
- Restriction enzymes (Ecor1, BAMH1, Sal1, Pst1) recognise the restriction site (small palindrome sequence - reads the same in both directions) and create a cut in a specific cleavage site of the palindrome sequence and generate Sticky and Blunt ends
- We cut our Plasmid Vector with the same restriction enzyme we used to cut the DNA of interest, the ends are compatible and can base-pair and then joined by DNA ligase - the plasmid is now called Recombinant DNA
NOTE:
When cleaving the gene of interest, other DNA fragments may be present in the solution due to incomplete purification of the insert.
Similarly, if the plasmid contains multiple restriction sites, it can also be cut into more than one piece, generating additional fragments.
As a result, during ligation, any compatible DNA fragment — not just the desired gene — may be inserted into the plasmid, leading to incorrect recombinant vectors.
- TRANSFORMATION occurs - recombinant vector introduced to host cell (bacteria) and bacteria is plated on a medium containing antibiotic
- Only transformed bacteria (those with the plasmid) survive when introduced to an antibiotic — because they have the antibiotic resistance gene.
- Among survivors, we then check which ones have the correct recombinant plasmid (sometimes using blue/white screening or colony PCR).
How will the bacteria now express the gene and produce the protein?
> Has a promoter sequence containing:
- transcriptional apparatus to bind to the promoter for transcription
- Transcription termination sequence
- Ribosome-binding site to enable translation —> produce protein
Note: Polylinker (or, multi cloning site), is a name given to an area with many restriction sites
- This is where our gene of interest would be, ligated onto, a plasmid
So we can:
- Express a protein for functional assays
- Put transformed bacteria into a culture flask and grow large amounts of it
- Then break them up and use some techniques to isolate and purify protein of interest in and use in assays
- Express a protein in a model organism:
Insert the recombinant plasmid into a, model, organism.,
+ Express, a,protein in,human cells, in,cell, culture:,
We, can even put the plasmid into human cells.,
- Describe the overall process of PCR, and the purpose of the reagents
- Denaturation
- heat briefly to seperate DNA molecules - Annealing
- cool to allow DNA primers (more stable) to form hydrogen bonds with ends of target sequence - Extension
- DNA polymerase (Taq polymerase - resistant to high temp, has proofreading and polymerase activity) adds nucleotides to the 3’ end of each primer
- List some of the uses of PCR as a technology
What can we use PCR for?
- Amplify and identify DNA from crime scene samples
- Amplify to identify the presence of waterborne pathogens
- Amplify to identify the presence of infectious pathogens
- Amplify to do DNA fingerprinting for identify/paternity testing
- Amplify to identify individuals in a population if animals
- Amplify to then clone into a plasmid
- Combine it with another technique to make RT-PCR, and identify the level of expression of a gene
