Nucleotides & Nucleic acids (lectures 15-18) Flashcards
Functions of DNA
1) genetic code
2) storage within the cell
3) accessibility for transcription
4) replication
5) meiosis
6) genome integrity
Components of nucleic acids
Heterocyclic base
Sugar (ribose or deoxyribose)
Phosphate
What is a nucleotide?
Base + sugar + phosphate
What is a nucleoside?
Base + sugar
What are the nucleosides in RNA?
Adenosine
Guanosine
Cytidine
Uridine
What are the nucleotides in RNA?
Adenylate
Guanylate
Cytidylate
Uridylate
What are the nucleosides in DNA?
Deoxyadenosine
Deoxyguanosine
Deoxycytidine
Thymidine
What are the nucleotides in DNA?
Deoxyadenylate
Deoxyguanylate
Deoxycytidylate
Thymidylate
Polymeric structure of DNA and RNA
DNA backbone is intrinsically negatively charged
• Easy to work with
• Wraps around positively charged histones = makes DNA more compact
• Prevents attack from nucleophiles
DNA > 100x more stable than RNA as the OH groups on the RNA lead to base-catalysed hydrolysis of the RNA backbone
• RNA needs to be more unstable to perform its function
• Want it all to naturally degrade
DNA = long term storage of information RNA = temporary transfer of information
What are Chargaff’s rules?
1) For a particular species: [A] = [T] & [G] = [C]
2) [A] + [T] / [G] + [C] varies between organisms
Watson-Crick model for DNA structure
2 antiparallel strands Right handed double helix Specific base pairing Bases stacked on the inside Backbone on the outside
Double helix dimensions
One turn of helix = 3.4 nm
Rise per base = 0.34 nm
10 base pairs per turn
Helix diameter = 2 nm
How is strength of base pairing different?
G-C has 3 hydrogen bonds so is slightly stronger
Sugar phosphate backbone
Negatively charged
Makes it difficult to pack tightly
Targeted for non-specific DNA binding
Binding by positively charged proteins
Why do we get major and minor grooves?
DNA molecule is asymmetrical
Backbones are closer together on one side leading to the minor groove
Further away on the other side leading to the major groove
Major groove
Information-rich
Sequence-specific DNA binding
‘Read’ the sequence without unwinding
Exploited by transcription factors
Minor groove
Information-poor
Infrequently used for sequence recognition
Binding typically alters DNA architecture
Single stranded nucleic acid structures
Always attempts to satisfy Watson-Crick base paring
RNA molecule manages to form a slight double helix due to palindromic sequences in the stem loop
How idid Meselson & Stahl prove DNA was semi-conservative using radioactive nitrogen isotopes?
Grew bacteria in nitrogen 15 containing medium (heavy nitrogen) so all DNA would be heavy DNA
Would then continue growing then in nitrogen 14 (light nitrogen)
Old strands = n15
New strands = n14
Density gradient equilibrium sedimentation
• Centrifugation
• As a molecule is centrifuged it will sediment when the buoyancy becomes equivalent to the centrifugal force
The more rounds of replication the more light fomrs of DNA
Will always have 2 intermediate species – one light strand & one heavy strand
DNA replication
Requires deoxyribonucleotide triphosphate (dNTP) precursors
The new DNA chain is assembled on a pre-existing DNA template
Requires a primer to begin synthesis
Mistake correction by the removal of mismatched nucleotides
• 3’-5’ exonuclease activity permits removal of incorrect nucleotides
• DNA polymerase travels 5-3 but it has the ability to reverse and & correct the mistake
Whats the different between exonucleases & endonucleases?
Exo digest from the end
Endo digest from the middle
Initiation of replication
Origin of replication (oriC)
One circular piece of DNA
DnaA is an oligomeric protein that can form rings or filaments
• Forms a 6 membered ring in bacteria
• 5 components of the ring bind to the 5 dnaA binding sequences
• AT rich tandem array of 13mers
• DnaA assembly stimulates unwinding of AT rich array
Unwinding the double helix
DnaB helicase – unwinds DNA
• Ring structure with a hole in the middle – the right size for ssDNA but too small for dsDNA
• It pulls the 2 strands apart
Recruited by dnaA
Loaded around ssDNA
ATPase-dependent translocation
Strand exclusion model
Stabalising ssDNA
Single stranded binding (SSB) protein
• Tetrameric protein
• ssDNA is wrapped around SSB tetramers
• Prevents secondary structure formation
Prepriming complex
DnaA recruits dnaB
DnaB begins to translocate 5’ –> 3’
Relaxing DNA molecules
Topoisomerase I Catalyses relaxation of supercoiled DNA 1) Cleavage of 1 strand 2) Passage of cut end under other strand 3) Resealing the break
Untangling DNA molecules
Topoisomerase II Catalyses untangling of DNA duplexes 1) Cleavage of both strands 2) Passage separate duplex molecule through break 3) Resealing the break
Primase
RNA primer – not DNA primer • RNA was formed earlier = ancestral artefact Required for the synthesis of DNA Provides initial free 3’ OH Primase can initiate but not extend
DnaG primase
• Synthesises RNA primer
• Recruited by dnaB
DNA polymerase III
Involved in DNA replication DNA polymerase core made up of: 1) Polymerase unit - alpha 2) Exonuclease domain - e 3) Sliding clamp - B2
What does the polymerase unit (alpha) do?
Has fingers, palm & thumb
5’ to 3’ DNA synthesis
Geometry enforces Watson-Crick base pairing
Adds one base onto the DNA strand
What does the exonuclease domain (e) do?
3’ to 5’ exonuclease
Proofreading
What does the sliding clamp (B2) do?
Beta subunit is a ring Hole big enough for dsDNA Allows polymerase III to stay in contact with the DNA strand at all times • Alpha subunit can be more efficient • Can produce lots of DNA quickly • Doesn't rely on diffusion events
The lagging strand problem
DNA polymerase synthesises 5’ to 3’
Lagging strand grows 3’ to 5’
Forms Okazaki fragments
Lagging strand loops back on itself so DNA polymerase can still move in the same direction
DNA polymerase III holoenzyme
DnaB recruits 2 DNA polymerases for coordinated synthesis of the leading and the lagging strand
The trombone model of the lagging strand
Looping of lagging strand
Lets go after adding 1000nts
New loop then formed
Loop lengthens & shortens like a trombone slide
Loop is short at the start of the Okazaki fragment
When finishing the fragment the loop is a lot bigger
Filling in the gaps in the lagging strand
DNA polymerase I • Fills in gaps between fragments • 5’ to 3’ exonuclease activity • Removes RNA primer & synthesises DNA • Slow (10nt/s)
DNA ligase
• Catalyses phosphodiester bond formation between 3’ hydroxyl and 5’ phosphate
• Fills in ‘nicks’ between Okazaki fragments
