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MBB266 > Rafferty (structure of NAs & their BPs) > Flashcards

Rafferty (structure of NAs & their BPs) Flashcards

1
Q

What do nucleotides polymerise to prod?

A
  • long chain of nucleic acids
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2
Q

How is the polarity of nucleotides defined?

A
  • where base attaches (to C1)
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3
Q

What is the difference between the structures of RNA and DNA?

A
  • in RNA hydroxyl attached to C2

- has big impact on structure

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4
Q

What are the diff H bonds poss in NAs and which are found in bases, and why?

A
  • 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
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5
Q

What does H bonding depend upon?

A
  • having approp groups
  • distance apart
  • angle
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6
Q

How long are H bonds?

A
  • typically 2.8Å to 3.2Å
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7
Q

Are H bonds linear and why?

A
  • need to be fairly linear, so bases also need to be linear
  • usually ∠30°deviation
  • if more then repulsion
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8
Q

What are the properties of purine bases?

A
  • 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
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9
Q

What are the properties of pyrimidine bases?

A
  • C, T and U
  • single 6 membered rings
  • all have 2 H bond acceptors and 1 donor
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10
Q

What is the only diff between thymine and uracil?

A
  • methyl group on thymine
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11
Q

Why does knowing the covalent structure of NAs not mean we know the 3D structure?

A
  • too many dof, so many ways structure could form

- 3D structure of each nucleotide determined by rotation about 7 conformational angles

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12
Q

What is the evidence for the Watson and Crick model of DNA?

A
  • microscopy and light scattering
  • Chargaff’s Rules
  • X-ray fibre diffraction
  • titration experiments
  • model building studies
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13
Q

How did microscopy and light scattering provide evidence for the Watson and Crick model of DNA?

A
  • 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
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14
Q

How did Chargaff’s Rules provide evidence for the Watson and Crick model of DNA?

A
  • looked at relative proportions of each base
  • amount of G≈C and A≈T
  • G/C = A/T ≈ 1
  • A + T ≠ G + C
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15
Q

How did X-ray diffraction provide evidence for the Watson and Crick model of DNA?

A
  • 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
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16
Q

How did titration experiments provide evidence for the Watson and Crick model of DNA?

A
  • 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
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17
Q

How did model building studies provide evidence for the Watson and Crick model of DNA?

A
  • built DNA models to try to explain X-ray fibre diffraction pattern
  • tried to incorp known info about stereochemistry of sugars, phosphates and bases
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18
Q

What were the features of the Watson and Crick model of DNA from their initial evidence?

A
  • 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)
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19
Q

How was H bonding an important part of the Watson and Crick model of DNA?

A
  • 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
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20
Q

What were the features of the final Watson and Crick model of DNA?

A
  • 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
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21
Q

What was the importance of Watson and Cricks work, apart from the structure of DNA?

A
  • 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
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22
Q

What are the diffs between A and B forms of DNA?

A
  • angles along backbone
  • in A bps tilted approx 20° to helix axis
  • A shorter and fatter (11bps in 28Å
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23
Q

What are the similarities between A and B forms of DNA?

A
  • right handed
  • anti parallel
  • WC bping
  • bases stacked
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24
Q

How easily can DNA switch between A and B forms, and why?

A
  • easily, w/o breaking bonds

- dynamic structure that can easily change in response to env

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25
Q

Where did early evidence for the structure of RNA come from?

A
  • ds RNA from retroviruses
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26
Q

What did diffraction patterns show about RNA structure, and why is this the case?

A
  • 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
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27
Q

What is a RNA/DNA duplex?

A
  • 1 strand of DNA complements 1 strand of DNA
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28
Q

What is the significance of conversion between A and B forms of DNA, and what evidence suggested this?

A
  • 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
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29
Q

What did fibre diffraction of DNA show?

A
  • 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
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30
Q

What did DNA crystallography show?

A
  • 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
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31
Q

What are the propeties of Z-DNA?

A
  • short GC repeating oligo
  • left handed double helix
  • 12 bps in 45Å
  • zgi zig backbone
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32
Q

What was discovered about Z-DNA in 2003 and 2005?

A

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)

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33
Q

What is the role of tRNA?

A
  • key adaptor molecule in protein synthesis
  • has anticodon that recognises 3 letter codon on mRNA
  • carries AAs ( prod nascent protein chain)
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34
Q

How many types of tRNA are there, and how do they work?

A
  • over 20
  • eg. Met TRNA
  • anticodon recognises codon on mRNA (eg, AUG for Met)
  • AA (eg. Met) attached to 3’ of tRNA
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35
Q

Why did the structure of tRNA take a long time to work out?

A
  • 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
36
Q

What is the “clover-leaf” structure of tRNA?

A
  • contains 4 short A form type helices = “stems”/”arms”

- T stem contains thymidine and pseudouridine (ψ)

37
Q

What is the L shaped representation of tRNA?

A
  • 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
38
Q

What are the main features of tRNA structure?

A
  • 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
39
Q

What are similarities/diffs between structures of tRNAs for diff AAs?

A
  • 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
40
Q

What are some examples of binding proteins that can interact w/ DNA or RNA

A
  • 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
41
Q

How can proteins recognise parts of DNA w/o unravelling it?

A
  • 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
42
Q

How do proteins interact w/ DNA?

A
  • generalised interactions w/ backbone

- specific interactions w/ bases in grooves of DNA

43
Q

What do we expect are the theoretical reqs for protein-DNA binding?

A
  • +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
44
Q

Are typical DNA seqs 2 fold, and why?

A
  • no 2 fold relationship between bases
  • only get true 2-fold if seq palindromic
  • ∴ 2 fold protein can only interact symmetrically w/ palindromic seqs
45
Q

How do prok repressors work?

A
  • obstruct RNA pol binding by binding to palindromic seqs overlapping w/ RNA pol binding site
  • no pol binding means no mRNA prod
46
Q

How do prok activators work?

A
  • bind palindromic sites and help RNA pol bind and start transcribing
47
Q

What were the first DNA binding proteins to have their structure solved, and how was this done?

A
  • 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”
48
Q

What specific interactions occur in a helix-turn-helix motif?

A
  • side chains on recognition helix
  • edges of bases in major groove -
    symmetrical (2 fold)
49
Q

What did the structures of protein-DNA complexes confirm about the role of helix-turn-helix motif?

A
  • recognition helices bind to major grooves of DNA

- DNA bent around molecule, to max interactions and emphasise flexibility of DNA

50
Q

How does protein structure change to max interactions w/ DNA?

A

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
51
Q

What is the zinc finger motif, and where is it found?

A
  • binding coord site for zinc ion, can be Cys2-His2 or Cys2-Cys2
  • often found in euk reg proteins
52
Q

What is the basic-leucine zipper motif?

A
  • 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
53
Q

How can DNA binding occur when not a helix?

A
  • 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
54
Q

What enzymes are w/in DNA binding proteins for cutting/copying/repairing DNA?

A
  • 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
55
Q

What is the MW and role of p53 suppressor?

A
  • 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
56
Q

What is the seq of p53?

A
  • 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
57
Q

Where are the majority of p53 mutations found, and what do they cause?

A
  • in core domain

- cause of approx 50% human cancers

58
Q

What critical contacts did the structure of p53 core domain complexed w/ DNA reveal?

A
  • 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
59
Q

What are some common mutations of p53?

A

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
60
Q

How do DNA-drug interactions play a role in some anti-cancer drugs?

A
  • 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
61
Q

How complex is tRNA compared w/ other RNA structures?

A
  • simple compared to some
62
Q

What are ribozymes?

A
  • enzymes made from RNA (not protein)
63
Q

What was the first eg. of a ribozyme found?

A
  • Tetrahymena, a protozoan
  • a self splicing piece of RNA
  • exon = mRNA expressed, converted into protein
  • intron = intervening seq, spliced out and not expressed
64
Q

What is the structure of ribozymes?

A
  • 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
65
Q

What is the structure of group II introns?

A
  • self splicing introns
  • prob ancestor of spliceosome
  • binds 2 Mg2+ via catalytic triad of bases and structurally important K+ (provides extra stability)
66
Q

How do RNA enzymes work?

A
  • 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”
67
Q

What are the characteristics of co-enzymes?

A
  • have nucleotide parts –> originally cofactors bound to ribozymes
  • have functional groups allowing interaction w/ bases in
68
Q

What are riboswitches, and what does this show?

A
  • 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
69
Q

What is the role of the ribosome?

A
  • cells protein synthesis machine
70
Q

What are the 2 subunits of ribosomes, and what are their sizes?

A

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)
71
Q

What does S mean in relation to ribosome size (30S/50S)?

A
  • measure of sedimentation rate, depends on frictional coefficients, shape, density
72
Q

What is the role of large subunit of ribosomes?

A
  • cat peptide bond formation

- 2 pieces of RNA

73
Q

How was ribosome structure studied?

A
  • 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
74
Q

Why did the predicted 2° and domain structures give little idea about the 3D structure of ribosomes?

A
  • 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
75
Q

What is the structure of the ribosome large subunit?

A
  • 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)
76
Q

What is the active site for protein synthesis, and how was this discovered?

A
  • long controversy over whether protein or RNA components that cat protein synthesis
  • active site located by soaking crystals w/ known inhibitors
  • defo all RNA
77
Q

What is the catalytic mechanism proposed for protein synthesis by ribosomes?

A
  • complex
  • involves crucial adenine in unusual 3D env v close to phosphate group
  • leads to abnormal protonations –> unusual activity
78
Q

What is the peptide exit tunnel in ribosomes and how is it visualised?

A
  • 1st suggested by EM
  • crystal structure confirmed existence
  • now seen in atomic detail leading from active site through molecule
  • tunnel bounded by RNA domains
79
Q

What is the role of the ribosome small unit?

A
  • decoding of genetic info during translation
  • by binding mRNA
  • subunit that binds tRNAs that read mRNA
80
Q

What is the structure of the ribosome small unit?

A
  • 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
81
Q

How do the subunits form whole ribosome?

A
  • 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
82
Q

Which parts of the ribosome are RNA?

A
  • the key parts
  • decoding centre (S subunit)
  • peptide tunnel
  • peptidyl active site
83
Q

What role do proteins have in the ribosome?

A
  • 2° role = stabilising structure
84
Q

How does the euk ribosome differ from the prok ribosome?

A
  • 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)
85
Q

How do many antibiotics work?

A
  • several classes directed against bacterial ribosomes

- most bind to RNA, not protein

86
Q

What are some examples of antibiotics which bind to ribosomal subunits and how do they work?

A
  • 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

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