Protein-Nucleic Acid Interactions - part 1Blackburn & Gait, Ch. 9

Define persistence length of nucleic acidKnow four forces used in protein-nucleic acid interaction and the details of eachKnow how DNA shape affects its ability to be bound by proteinsKnow other geometric constraints of dsDNAKnow geometric constraint of ss nucleic acidsUnderstand how proteins interact nonspecifically with DNA & RNA

HU proteingene-5 from phageRNase A (some slight specificity)DNase Ipolymerases (E.coli pol and HIV RT)

Protein-Nucleic Acid Interactions

Possible forces used:Electrostatic, Dipolar (H-bonds), Hydrophobic, Dispersion (stacking)

Persistence length:Length of DNA (RNA) that remains rodlike in its configuration

At 100 mM NaCl, persistence length of DNA is ~150 bp

Remember: ~1 meter of DNA in a human cell (diameter of cell ~10-5 m)Therefore DNA is highly compacted in nucleosome (chromatin)

How does complexation with proteins cause DNA bending?

Protein-Nucleic Acid Interactions

Forces used:Electrostatic, Dipolar (H-bonds), Hydrophobic, Dispersion (stacking)

Electrostatic• salt bridges (~40 kJ/mol of stabilization per salt bridge)• negative phosphates (DNA/RNA) with positive AA (Lys, Arg, His)• influenced by [salt]; as [salt] strength of salt bridge • pattern of salt bridges can distinguish between ss and ds DNA/RNA and between B-DNA and Z-DNA• overall electrostatic field of protein can orient polyanionic nucleic acid, modeling of interactions

Protein-Nucleic Acid Interactions

Forces used:Electrostatic, Dipolar (H-bonds), Hydrophobic, Dispersion (stacking)

Electrostatic

Protein-Nucleic Acid Interactions

Forces used:Electrostatic, Dipolar (H-bonds), Hydrophobic, Dispersion (stacking)

Dipolar forces (H-bonds)• between AA side chains (as well as backbone amides and carbonyls) and Nucleic acid bases and sugar phosphate oxygens• water mediated

X-H ••• Y-R

+ + - -

Hydrophobic (entropic forces)• layer of water around protein or DNA• when pro and NA interact, ordered water at interface are released ( entropy) • hydrophobics on inside, hydrophillics on outside

Dispersion forces (base stacking)• hydrophobic interactions and dispersion• molecules with no net dipole can attract each other by a transient dipole-induced dipole effect (London)• dispersion forces important for base stacking and also for ss regions because aromatic side chains of protein intercalate between bases in ss nucleic acids

Protein-Nucleic Acid Interactions

Geometric Constraints

DNA shape - AT sequences/flexibilityRNA single strand - Hoogsteen pairs, triples, bulges

Protein-Nucleic Acid Interactions

Geometric ConstraintsdsDNAhigh (-) charge; protein domains that interact with it have a complementary (+) surface, polar or charged side chains (used a lot by T.F.) interact with phosphate oxygens (backbone) Seq-specific - repressor operator complexNonseq-specific - DNA polymerase I 3’5’ exonuclease

ds B-DNAAnti-parallel -ribbon (protein) interacts with minor groove; H-bonds to phosphates

Protein-Nucleic Acid Interactions

Geometric Constraintsds B-DNA-helix (protein) interacts in major groove with basesmost common because of H-bond donors and acceptors in MAJOR groove; interaction involves at least 1 H-bond

Protein-Nucleic Acid Interactions

Geometric Constraintsss Nucleic AcidsHydrophobic bases exposedSSBP will have more phobic NA binding surface

RNA - lots of regions of single-strandedness (bulges, loops, pseudoknots)A-form MAJOR groove deeper and bases more inaccessibleBulges can help widen MAJOR grooveLoop structures can bind proteins

Protein-Nucleic Acid Interactions

Geometric Constraintsss Nucleic AcidsRNA - lots of regions of single-strandedness (bulges, loops, pseudoknots)Loop structures can bind proteins

Protein-Nucleic Acid Interactions

Non-specific InteractionsPackagingEuks - nucleosomeProks - similar protein to histones that is small & basicNo nucleosome structure formedCrystal structure of E.Coli DNA binding protein II (HU)

• Binds DNA by encircling it• Extended -sheet arms contact DNA• DNA binding ability increases as #basic AAs increase• protein wedges polymerize thus inducing DNA to form supercoiled structures• HU binds ss or dsDNA so must contact sugar-phosphate backbone

Protein-Nucleic Acid Interactions

Non-specific Interactionsss nucleic acid binding proteinsDuring DNA replication in phage fd, gene-5 protein binds to ssLots of -strandsLys/Arg neutralize phosphate backbone and bases stack against aromatic amino acid side chains

Protein-Nucleic Acid Interactions

Non-specific InteractionsExonucleases & endonucleasesRNase A cleaves RNA but also binds ssDNA (competitive inhibitor)Group of (+) charged residues on protein (anion binding site)Electrostatics are main force involved so little seq-specificity Specific for sequence with pyr 5’ to cleavage site since Thr45 H-bonds

Protein-Nucleic Acid Interactions

Non-specific InteractionsExonucleases & endonucleasesDNase I - forces (VDW contacts, H-bonds, salt bridges)cleaves dsDNALittle seq-specificityStructure of complex - exposed loop of enzyme binds in minor groove of DNA which mimics nicked DNA

Protein-Nucleic Acid Interactions

Non-specific InteractionsExonucleases & endonucleasesDNase I - forces (VDW contacts, H-bonds, salt bridges)Little seq-specificity

Protein-Nucleic Acid Interactions

Non-specific InteractionsPolymerasesDNA-dependent DNA polymerase: E.Coli DNA pol I and IIIPol III

• chief replicative subunitPol I

• repairs damaged DNA and converts Okazaki fragments into complete genomic DNA• large fragment (Klenow) - DNA pol, 3’-5’ exo• small subunit - 5’-3’ exo

3’-5’ exo

DNA pol

Catalytic AA •(Asp705, Asp882, Glu883)

Protein-Nucleic Acid Interactions

Non-specific InteractionsPolymerasesDNA-dependent DNA polymerase: E.Coli DNA pol I and IIIPol I - divalent cations important, may bind to catalytic AAsCation 1 - positions OH- to attack phosphorus and generate pentacovalent transition stateCation 2 - stabilizes leaving group