DNA Flashcards
Define: Genome Gene Nucleotide Sugar phosphate backbone
Genome: all genetic information of an organism
Gene: the basic unit of inheritance within a chromosome, a sequence for a protein or RNA
Nucleotide: made of a phosphate sugar and a base
SPB: runs along the DNA as a constant backbone whilst the bases can change between A T C and G
What are nucleotides and nucleosides?
- subunits of nucleic acids
Nitrogen-containing base, five-carbon sugar and one or more phosphate groups - nucleosides have no phosphate group
Which sugar is found in DNA vs that found in RNA?
DNA: deoxyribose (H on the 2’ carbon)
RNA: ribose (OH on 2’ carbon)
What are the 4 bases?
Adenine, thymine (uracil in RNA), cytosine and glycine
What are purines?
A + G
Nitrogen-rich heterocyclic double-ring structures
What are pyrimidines?
C U + T
Nitrogen-rich heterocyclic single ring structures
How can bases be modified or damaged? Exmaples
Methylation
UV light and carcinogens
How are nucleic acids formed? What are their general properties?
Nucleotides are joined together by a phosphodiester linkage between 5’ and 3’ carbon atoms to form nucleic acids
Each sequence has a 5’ and 3’ end
Backbone is negatively charged
Bases can link to strands of DNA through hydrogen bonds
What is the difference between the link between AT and GC
A + T pair with 2 hydrogen bonds
G + C pair with 3 hydrogen bonds
The structures of the pairs are similar and so they are all the same distance apart along the DNA strands. This would not be the case if you paired the wrong bases
What is X-ray diffraction?
A scatter of x-rays can form an image of DNA and show its helical structure and size
What are minor and major grooves?
On the major groove side, the DNA backbone molecules are further apart.
In the minor groove side, the DNA backbone molecules are closer together.
How does DNA length affect its flexibility?
Long DNA is more flexible than short DNA
How is short DNA less flexible?
Electrostatic repulsion of phosphates (bending brings phosphates closer together and their electrostatic repulsion pushes them apart again)
Compressive base stacking (bending is hard because its energetically favourable that bases ate stacked up perfectly)
What is the persistence length of DNA?
The length along which a thermally excited bend of 1 radian typically occurs. If a DNA strand is beloved this length is will be straight
How can proteins interact with DNA?
- recognise and bind t specific DNA sequences
- recognise features such as DNA damage
- bind to DNA non-specifically
What does semi-conservative replication mean?
In the new DNA, one strand is from the parent DNA (template) and one is a newly replicated strand
What are the domains of the DNA polymerase core enzyme fold? What are their functions?
Fingers domain and thumb domain are to grip the DNA
Palm domain is where the active region in catalysis lays
What are the main properties of DNA polymerase III? (4)
- main replicating enzyme in E.coli
- 9 protein subunits
- 3’-5’ exonuclease activity with proofreading
- mg2+ cofactor
Which end of the DNA or RNA molecule does DNA polymerase add nucleotides to?
3’
binds to a primer with a free 3’ hydroxyl group
What are oriC and Ter?
Replication origin and terminator sequence of E.coli genome
How does replication start in E.coli?
Specific oriC sequences are recognised and bound by an initiator.
A+T rich regions surrounding oriC are easily unwound and strand separation begins via DNA helicase
What does DNA helicase do?
Unwinds double stranded DNA to produce single-stranded DNA for replication using energy from ATP
What is primase?
A specialised RNA polymerase that make new short RNA primers that provide the 3’-hydroxyl group that DNA polymerase needs to start DNA synthesis
What is the bi-directional replication fork?
In circular DNA, the two replication forks start moving from initiation in opposite directions
What is DNA polymerisation?
DNA polymerase adds nucleotides (dNTPs) to the 3’ end of the growing chain.
What is DNA polymerisation?
DNA polymerase adds nucleotides (dNTPs) to the 3’ end of the growing chain.
What is DNA polymerisation?
DNA polymerase adds nucleotides (dNTPs) to the 3’ end of the growing chain.
What is DNA polymerisation?
DNA polymerase adds nucleotides (dNTPs) to the 3’ end of the growing chain.
What does the catalytic site of DNA polymerase II require to work?
Mg2+ helps stabilise the phosphates and activate OH
Aspartates co-ordinate the metal ions
Triphosphate provides a high energy bond
What and how does DNA polymerase proofread?
Weak hydrogen bonds can form inbetween incorrect bases distorting the DNA structure
Polyerase recognises this and it uses its exonuclease activity to remove the base from 3’-5’
Define exonuclease
an enzyme that cleaves nucleotides one at a time from the ends of polynucleotides
Define endonuclease
an enxyme that cleaves nucleotides from within polynucleotides
What is discontinuous DNA replication?
The polymerase can only move from 5’-3’.
On one strand this is easy and the enzyme can move continuously but on the other, the strand is unwinding from behind the polymerase so it must constantly restart creating Okazaki fragments with many primase primers
How are Okazaki fragments connected?
- RNA primers removed and replaced by DNA
- 3’-5’ proofreading
- DNA ligase catalyses the phosphate linkage
How is replication terminated in E.coli?
DNA polymerase reached a Ter sequence
Tus binds to Ter sites and provides a physical block to the replication fork.
Different Ter groups face in different directions and the right one must be bound to or else the polymerase will push past it
Name 5 types of DNA repair
Direct repair (specific base damage) Mismatch repair (incorrect base pairing) Base excision repair (damaged bases) Nucleotide excision repair (bulky DNA damage) Homologous Recombination (dsDNA breaks)
What is the structure of RNA?
- same as DNA but uracil rather than thymine
- ribose in place of deoxyribose
- usually single stranded
- can form secondary structures like hairpins and bulges
How can RNA tertiary structures form?
What is the point of this?
- hairpin structures can fold together into tertiary structures
- this can provide more structural and catalytic roles
What is transcription?
Double stranded DNA is unwound and one of the strands is copied into RNA from 5’-3’
What is a transcription bubble?
A small section of unwound DNA from which the template strand is read
What is the structure of e.coli RNA polymerase?
A large complex made of 5 subunits Wh
What is the structure of e.coli RNA polymerase?
A large complex made of 5 subunits Wh
What is the function of RNA polymerase?
- unwinds DNA and allows for the rewinding after RNA has been synthesised
- free nucleotides come into the molecule through the ribonucleoside triphosphate uptake channel
- RNA leaves the molecule through the RNA exit channel
What are the three stages of the transcription cycle?
Initiation, elongation and tremination
What happens in the initiation phase of transcripton?
RNA polymerase binds to DNA
A transcription bubble is formed
RNA synthesis starts
What happens in the elongation phase of transcription?
The RNA polymerase moves aloong the DNA, producing an RNA strand behind it
What happens in the termination phase of transcription?
DNA and RNA are released and RNA polymerase can be used again
What are bacterial promoter sequences?
- sequences that dictate where transcription starts
- contain the -10 position (prinbnow box) and the -35 position that are reocgnised by sigma factors bound to RNA polymerase
What is the consensus sequence and how does it affect bacterial promoter sequences?
- the most optimal sequence for a specific sigma factor
- the closer the -35 and -10 sequences are to their consensus the stronger the promoter
What is Rho-dependent transcription termination?
Rho protein binds to rut sites and uses energy from ATP hydrolysis to move along the RNA to catch up with it and pull it out of the RNA polymerase
What is Rho-independent transcription termination?
Specific sequences with complementary sequences fold up to form hairpin structures that stop transcription
What are e.coli transcription inhibitors?
Drugs that stop e.coli polymerase from working whilst keeping human RNA unaffected
