Rafferty (structure of NAs & their BPs) Flashcards
What do nucleotides polymerise to prod?
- long chain of nucleic acids
How is the polarity of nucleotides defined?
- where base attaches (to C1)
What is the difference between the structures of RNA and DNA?
- in RNA hydroxyl attached to C2
- has big impact on structure
What are the diff H bonds poss in NAs and which are found in bases, and why?
- N-H - - - - > O
- N-H - - - - > N
- O-H - - - - > O
- O-H - - - - > N
- only N-H ones found in bases as no hydroxyl groups found in bases
What does H bonding depend upon?
- having approp groups
- distance apart
- angle
How long are H bonds?
- typically 2.8Å to 3.2Å
Are H bonds linear and why?
- need to be fairly linear, so bases also need to be linear
- usually ∠30°deviation
- if more then repulsion
What are the properties of purine bases?
- A and G
- double rings (one 5 and one 6 membered)
- don’t H bond in normal structure of bases, but are capable of it
- adenine has donor and acceptor for H bonding
- guanine has 2 donors and 1 acceptor
What are the properties of pyrimidine bases?
- C, T and U
- single 6 membered rings
- all have 2 H bond acceptors and 1 donor
What is the only diff between thymine and uracil?
- methyl group on thymine
Why does knowing the covalent structure of NAs not mean we know the 3D structure?
- too many dof, so many ways structure could form
- 3D structure of each nucleotide determined by rotation about 7 conformational angles
What is the evidence for the Watson and Crick model of DNA?
- microscopy and light scattering
- Chargaff’s Rules
- X-ray fibre diffraction
- titration experiments
- model building studies
How did microscopy and light scattering provide evidence for the Watson and Crick model of DNA?
- DNA too small to use X-rays as no lenses exist and no good focussing methods
- so use EM –> showed DNA long thin molecule approx 20Å in diameter
- light scattering = some light hits protein/NA and is scattered, vary wavelength and measure scattering at diff angles –> showed DNA long thin rod shaped molecule
How did Chargaff’s Rules provide evidence for the Watson and Crick model of DNA?
- looked at relative proportions of each base
- amount of G≈C and A≈T
- G/C = A/T ≈ 1
- A + T ≠ G + C
How did X-ray diffraction provide evidence for the Watson and Crick model of DNA?
- even w/o lenses can still deduce a lot
Astbury: - regular structure
- 3.4Å repeating unit along fibre
- suggested bases like “pile of pennies”
Franklin & Wilkins: - put DNA in controlled humidity chambers
- found 2 forms –> B-form simpler blurred pattern and A form sharp diffraction pattern and gave lots of info
Watson & Crick: - interested in B form
- showed double diamond pattern was helix
- big distance on diffraction pattern = small distance in reality
- pattern suggested helix of 34Å pitch w/ 10 small repeating units
How did titration experiments provide evidence for the Watson and Crick model of DNA?
- if look at indiv nucleotides in solution can titrate phosphate groups at pH 2 and bases at pH 4.5
- in DNA phosphates can’t be titrated and bases cannot be titrated
How did model building studies provide evidence for the Watson and Crick model of DNA?
- built DNA models to try to explain X-ray fibre diffraction pattern
- tried to incorp known info about stereochemistry of sugars, phosphates and bases
What were the features of the Watson and Crick model of DNA from their initial evidence?
- helical structure (from diffraction pattern)
- base stacking (from 3.4Å repeat in diffraction pattern)
- 2 chains (inferred from density measurements and features of X-ray pattern)
- regular sugar-phosphate backbone (X ray patterns same from diff species and doesn’t depend on base composition)
How was H bonding an important part of the Watson and Crick model of DNA?
- said bases prob H bonded to each other, s titrations showed bases buried from water
- but H bonds only strong if linear arrangement of donor-H-acceptor
- only poss if H bonding between bases in diff chains
- also backbone must be regular and unaffected by base composition
- purine to purine would make backbones distant and pyrimidine to pyrimidine would make backbones close –> must be purine to pyrimidine to make it regular
- found 2 H bonds between AT and 3 between GC (explains Chargaff’s Rules)
- freely interchangeable fit of A=T, T=A, G≡C and C≡G into 2 chains running in opp directions
- further evidence from thermal denaturation of DNA –> when DNA heated eventually “melts” and loses structure, the more GC, the more stable and higher the melting temp, due to more H bonds
What were the features of the final Watson and Crick model of DNA?
- sugar-phosphate backbone on outside
- bps stacked on inside
- double, right handed, anti-parallel helix
- major and minor grooves
- 10bps per turn
- bases carry genetic info and backbone has structural role
What was the importance of Watson and Cricks work, apart from the structure of DNA?
- also immediately realised biological implications
- structure suggested mechanism for storing and rep genetic info
- 1 strand (template) carries genetic info and other complementary to it
What are the diffs between A and B forms of DNA?
- angles along backbone
- in A bps tilted approx 20° to helix axis
- A shorter and fatter (11bps in 28Å
What are the similarities between A and B forms of DNA?
- right handed
- anti parallel
- WC bping
- bases stacked
How easily can DNA switch between A and B forms, and why?
- easily, w/o breaking bonds
- dynamic structure that can easily change in response to env
Where did early evidence for the structure of RNA come from?
- ds RNA from retroviruses
What did diffraction patterns show about RNA structure, and why is this the case?
- only A form (never B)
- in B form OH would clash w/ O in adjacent phosphate and bases
- in A form backbone angles diff so phosphate groups further away and base tilted 20° out of way
What is a RNA/DNA duplex?
- 1 strand of DNA complements 1 strand of DNA
What is the significance of conversion between A and B forms of DNA, and what evidence suggested this?
- only A form in all diffraction experiments and RNA can only be A form
- suggests A form can from DNA/RNA duplex, for transcrip
- and B form cannot form duplex, can only pair w/ other DNA strand, so used for rep
What did fibre diffraction of DNA show?
- in fibre long molecules of DNA w/ diff seqs roughly aligned on fibre axis
- poor diffraction pattern, not enough to solve structure w/o other evidence
What did DNA crystallography show?
- computationally based analysis instead of direct visualisation w/ lenses
- millions of short DNA oligos, all identical seq and perfectly aligned in crystal
- v detailed yet simple diffraction pattern, mathematically interpretable
- 3D image showed positions of atoms directly w/ no ambiguity
What are the propeties of Z-DNA?
- short GC repeating oligo
- left handed double helix
- 12 bps in 45Å
- zgi zig backbone
What was discovered about Z-DNA in 2003 and 2005?
2003:
- many Z-DNA and Z-RNA binding proteins identified, inc ones involved in tumour response and viral pathogenicity
- full sig still unclear bu tregions suggested to facilitate DNA unwinding (by destabilising) or supercoiling
2005:
- B-form can transition into Z-form (could happen in genome)
What is the role of tRNA?
- key adaptor molecule in protein synthesis
- has anticodon that recognises 3 letter codon on mRNA
- carries AAs ( prod nascent protein chain)
How many types of tRNA are there, and how do they work?
- over 20
- eg. Met TRNA
- anticodon recognises codon on mRNA (eg, AUG for Met)
- AA (eg. Met) attached to 3’ of tRNA
Why did the structure of tRNA take a long time to work out?
- parts of seq complementary but written back to front –> parts may bp w/in itself (ds stems and intervening loops)
- larger variety of bases in RNA –> eg. inosine (often found at 1st (5’) position in anticodon loop to enable some “wobble” in pairing w/ 3rd base (3’) in mRNA
What is the “clover-leaf” structure of tRNA?
- contains 4 short A form type helices = “stems”/”arms”
- T stem contains thymidine and pseudouridine (ψ)
What is the L shaped representation of tRNA?
- more realistic representation of 2D structure
- T stem and AA stem = 1 A form type helix
- D stem and AC stem = another A form type helix
What are the main features of tRNA structure?
- each stem resembles A-DNA structure
- AA binding site at 1 extreme end of structure (3’), anticodon triplet at other end of structure and T loop (ribosome binding) at 3rd corner of L shaped structure –> separating functional sites so no interference is highly efficient structural design
- 55% bases paired in W-C manner –> unpaired bases in D stem and anticodon end of AC stem
- some unusual base triplets
- 95% bases stacked on top of each other –> shows this is major stabilising force, edges of bases rich in O and N so want to H bond to water or each other, faces of bases aromatic rings so interact unfavourably w/ water and stack on top of each other (attracted by VdW) to avoid contact w/ water
What are similarities/diffs between structures of tRNAs for diff AAs?
- same structural principles for all
- 3 key areas in same relative orientations, but other parts can vary
- other parts recognised by cognate tRNA synthetase that attaches AA
What are some examples of binding proteins that can interact w/ DNA or RNA
- proteins that reg gene expression
- proteins involved in DNA packaging
- restriction enzymes that cut DNA at specific seqs
- pols that copy DNA/RNA to new DNA/RNA seqs
- proteins that repair DNA
- DNA unwinding proteins that stabilise ss DNA by preventing duplex formation
How can proteins recognise parts of DNA w/o unravelling it?
- proteins can detect distortions in backbone
- edges of bases can be read easily in major groove
- can also be read in minor groove but access more difficult
How do proteins interact w/ DNA?
- generalised interactions w/ backbone
- specific interactions w/ bases in grooves of DNA
What do we expect are the theoretical reqs for protein-DNA binding?
- +ve charged sidechains interact w/ -ve phosphate groups = non-specific binding
- protein sidechains could “read” edges of bases = specific binding (A=T has acceptor where G≡C has donor, allowing discrimination of bases w/o unfolding DNA
- complementarity of structures –> shape of protein should match that of DNA to max interactions
- symmetry –> dimeric proteins have 2 fold symmetry, as does DNA backbone, ∴ have symmetrical interactions w/ each other through compatibility of structures
Are typical DNA seqs 2 fold, and why?
- no 2 fold relationship between bases
- only get true 2-fold if seq palindromic
- ∴ 2 fold protein can only interact symmetrically w/ palindromic seqs
How do prok repressors work?
- obstruct RNA pol binding by binding to palindromic seqs overlapping w/ RNA pol binding site
- no pol binding means no mRNA prod
How do prok activators work?
- bind palindromic sites and help RNA pol bind and start transcribing
What were the first DNA binding proteins to have their structure solved, and how was this done?
- overexpressed and solved by X-ray crystallography
- bacterial gene regulatory proteins
- -> E. Coli CAP (activates genes in cAMP presence)
- -> CRO repressor (regulatory protein in bacteriophage λ)
- -> λ repressor (regulatory protein in bacteriophage λ)
- all quite diff structures but all dimers and have common DNA binding motif feature = “helix-turn-helix motif”
What specific interactions occur in a helix-turn-helix motif?
- side chains on recognition helix
- edges of bases in major groove -
symmetrical (2 fold)
What did the structures of protein-DNA complexes confirm about the role of helix-turn-helix motif?
- recognition helices bind to major grooves of DNA
- DNA bent around molecule, to max interactions and emphasise flexibility of DNA
How does protein structure change to max interactions w/ DNA?
X-ray structures of trp repressor solved w/ and w/o Trp bound:
- when Trp bound (high Trp conc)
- -> DNA recognition helices 34Å apart
- -> repressor binds to DNA
- -> RNA pol binding blocked
- -> no mRNA made for Trp biosynthesis operon enzymes
- -> Trp synthesis stops
- no Trp bound (low Trp conc)
- -> diff protein conformation w/ helices only 26Å apart
- -> cannot bind DNA
- -> RNA pol binds
- -> mRNA can now be made for biosynthetic enzymes
- -> Trp synthesis starts
What is the zinc finger motif, and where is it found?
- binding coord site for zinc ion, can be Cys2-His2 or Cys2-Cys2
- often found in euk reg proteins
What is the basic-leucine zipper motif?
- can be 1 continuous helix or chain can double back on self in loop
- zipper also used as dimerisation motif in non-DNA binding proteins
How can DNA binding occur when not a helix?
- β-ribbons formed from 2 β-strands can be used
- β-ribbon fits snugly in major groove of DNA
- sidechains on β-strands interact w/ edges of bases
- alt, non-specific contacts can be made by edge of β-strand in minor groove when packaging DNA
What enzymes are w/in DNA binding proteins for cutting/copying/repairing DNA?
- endonuclease EcoRI = cleaves palindromic seq, ∴ dimer, complementary structure “embraces” DNA
- DNA pol I = cat step by step formation of new DNA strand on template strand, deals w/ all DNA seqs not just palindromic, ∴ monomer, large circular cleft wraps round DNA, complementary structure like a “hand” grasping DNA
What is the MW and role of p53 suppressor?
- MW = 53000
- preserves integrity of genome during cell division
- if DNA damaged stalls cell cycle until repairs made, and if damage too extreme then programmed cell death
What is the seq of p53?
- approx 400 residues and 3 functional domains
- activation domain (N-ter) = can activate transcrip of genes
- core domain = DNA binding to specific seq
- tetramerisation domain = makes 4 p53s come together, mol assembly recognises 4 target DNA seq separated by 0-13 bps
Where are the majority of p53 mutations found, and what do they cause?
- in core domain
- cause of approx 50% human cancers
What critical contacts did the structure of p53 core domain complexed w/ DNA reveal?
- Zn2+ stabilises DNA binding loop
- Arg248 contacts backbone in minor groove (mutated in around 10% human cancer causing mutations)
- DNA ≈B form but wider major goove
- strand-loop-helix motif in major groove
What are some common mutations of p53?
6 most common mainly affect non specific DNA binding (account for ≈40% p53 derived tumours):
- 2 arginines –> sugar-phosphate backbone
- 3 arginines –> H bond w/in protein structure and stabilise protein conformation
- Gly245 stabilises structure of loop in minor groove
Other 60% are mutations of residues close to protein-DNA interface
- disrupt seq-specific interactions
- proteins no longer recognise correct DNA seq
How do DNA-drug interactions play a role in some anti-cancer drugs?
- block DNA rep
- some studied by single crystal X-ray diffraction –> intercalating drugs, major groove binding drugs, minor groove binding drugs
- drugs may work by disrupting DNA-protein interactions and preventing transcrip
How complex is tRNA compared w/ other RNA structures?
- simple compared to some
What are ribozymes?
- enzymes made from RNA (not protein)
What was the first eg. of a ribozyme found?
- Tetrahymena, a protozoan
- a self splicing piece of RNA
- exon = mRNA expressed, converted into protein
- intron = intervening seq, spliced out and not expressed
What is the structure of ribozymes?
- similar structural principles to tRNAs
- majority of bases form WC bps in A form double helices
- almost all bases stacked on others
- base triplets/metal ions etc. link bits of structure together
- like RNA as specific H bonds form 3D structure and base stacking of hydrophobic surfaces drives folding
What is the structure of group II introns?
- self splicing introns
- prob ancestor of spliceosome
- binds 2 Mg2+ via catalytic triad of bases and structurally important K+ (provides extra stability)
How do RNA enzymes work?
- specifically bind substrate
- -> unpaired RNA bases have ability to specifically bind complementary seq in RNA/DNA structure
- -> most ribozymes cat reactions involving NA substrates
- groups w/ unusual activity that can cat reactions
- -> only 4 bases gives much less scope for unusual reactivity than proteins
- -> bound metal ions may give unusual reactivity
- -> nucleobases in unusual 3D envs may create unusual chem properties
- protein enzymes carry out most cellular reactions now
- ribozymes thought to be remainder of early “RNA world”
What are the characteristics of co-enzymes?
- have nucleotide parts –> originally cofactors bound to ribozymes
- have functional groups allowing interaction w/ bases in
What are riboswitches, and what does this show?
- mRNA segments that can fold up and bind small target molecules and affect mRNA transcrip
- demonstrates RNA molecule can reg gene synthesis
- show they can bind small molecules (AAs/cofactors)
- could be descended from RNA-world pre-protein regulatory systems
- pot antibiotic targets
- mainly in bacteria but some euk riboswitches recently discovered in plants and fungi
What is the role of the ribosome?
- cells protein synthesis machine
What are the 2 subunits of ribosomes, and what are their sizes?
Large subunit: (50S)
- approx 1.7 Md
- 2 pieces RNA = 2900 and 120 bases
- 34 proteins (L1-L34)
Small subunit: (30S)
- approx 1.0Md
- 1 piece RNA = 1500 bases
- 21 proteins (S1-S21)
What does S mean in relation to ribosome size (30S/50S)?
- measure of sedimentation rate, depends on frictional coefficients, shape, density
What is the role of large subunit of ribosomes?
- cat peptide bond formation
- 2 pieces of RNA
How was ribosome structure studied?
- EM = better resolution allowed to distinguish between L and S subunits
- neutron scattering = isotopically label pairs of proteins, reassemble ribosomes, then carry out neutron scattering for lots of pairs and triangulate distances
Why did the predicted 2° and domain structures give little idea about the 3D structure of ribosomes?
- 3D structure shows complicated organisation of RNA helices w/ clear domain organisations and proteins embedded on periphery
- peptidyl transfer active site w/ inhibitor bound in centre
What is the structure of the ribosome large subunit?
- active site cleft has no proteins v close to it apart from N-ter/C-ter of some
- core of subunit tightly packed mass of RNA helices
- proteins on surface stabilise interactions between RNA domains
- 3D structure more complex than 2D suggests (complex 3D jigsaw of RNA forms ribosome core)
What is the active site for protein synthesis, and how was this discovered?
- long controversy over whether protein or RNA components that cat protein synthesis
- active site located by soaking crystals w/ known inhibitors
- defo all RNA
What is the catalytic mechanism proposed for protein synthesis by ribosomes?
- complex
- involves crucial adenine in unusual 3D env v close to phosphate group
- leads to abnormal protonations –> unusual activity
What is the peptide exit tunnel in ribosomes and how is it visualised?
- 1st suggested by EM
- crystal structure confirmed existence
- now seen in atomic detail leading from active site through molecule
- tunnel bounded by RNA domains
What is the role of the ribosome small unit?
- decoding of genetic info during translation
- by binding mRNA
- subunit that binds tRNAs that read mRNA
What is the structure of the ribosome small unit?
- still big
- 96% nucleotides identified and all 20 proteins
- complex 3D jigsaw of RNA domains –> WC bps and lots of stacking
- proteins on outside, link RNA domains
- decoding centre entirely RNA
How do the subunits form whole ribosome?
- small subunit can rotate 12° relative to large subunit
- may relate to how ribosome moves along mRNA whilst tRNAs enter and leave
- mRNA fits through by twisting subunits relative to each other
Which parts of the ribosome are RNA?
- the key parts
- decoding centre (S subunit)
- peptide tunnel
- peptidyl active site
What role do proteins have in the ribosome?
- 2° role = stabilising structure
How does the euk ribosome differ from the prok ribosome?
- larger and more complex translation process
- euk-specific elements considerably expand network of interactions w/in ribosome
- key rRNA molecules closely related in seq (esp in active site)
How do many antibiotics work?
- several classes directed against bacterial ribosomes
- most bind to RNA, not protein
What are some examples of antibiotics which bind to ribosomal subunits and how do they work?
- tetracycline (S) = binds to tRNA A site
- streptomycin (S) = interferes w/ mRNA/tRNA recognition (error prone)
- chloramphenicol (L) = blocks tRNA assoc w/ A site
- erythromycin (L) = blocks entrance to tunnel
