Protein-Nucleic Acid Interactions - part 1 Blackburn & Gait, Ch. 9 Neidle, Ch. 7 Possible forces used: Define persistence length of nucleic acid Know four forces used in protein-nucleic acid interaction and the details of each Know how DNA shape affects its ability to be bound by proteins Know other geometric constraints of dsDNA Know geometric constraint of ss nucleic acids Understand how proteins interact nonspecifically with DNA & RNA HU protein gene-5 from phage RNase A (some slight specificity) DNase I polymerases (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/flexibility RNA single strand - Hoogsteen pairs, triples, bulges

Protein-Nucleic Acid Interactions Geometric Constraints DNA shape - AT sequences/flexibility

Protein-Nucleic Acid Interactions Geometric Constraints dsDNA high (-) 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 complex Nonseq-specific - DNA polymerase I 3’  5’ exonuclease ds B-DNA Anti-parallel  -ribbon (protein) interacts with minor groove; H-bonds to phosphates

Protein-Nucleic Acid Interactions Geometric Constraints ds B-DNA  -helix (protein) interacts in major groove with bases most common because of H-bond donors and acceptors in MAJOR groove; interaction involves at least 1 H-bond

Protein-Nucleic Acid Interactions Geometric Constraints ss Nucleic Acids Hydrophobic bases exposed SSBP 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 inaccessible Bulges can help widen MAJOR groove Loop structures can bind proteins

Protein-Nucleic Acid Interactions Geometric Constraints ss Nucleic Acids RNA - lots of regions of single-strandedness (bulges, loops, pseudoknots) Loop structures can bind proteins

Protein-Nucleic Acid Interactions Non-specific Interactions Packaging Euks - nucleosome Proks - similar protein to histones that is small & basic No nucleosome structure formed Only protein crystallized is 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 Interactions ss nucleic acid binding proteins During DNA replication in phage fd, gene-5 protein binds to ss Lots of b-strands Lys/Arg neutralize phosphate backbone and bases stack against aromatic amino acid side chains

Protein-Nucleic Acid Interactions Non-specific Interactions Exonucleases & endonucleases RNase 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 Interactions Exonucleases & endonucleases DNase I - forces (VDW contacts, H-bonds, salt bridges) cleaves dsDNA Little seq-specificity Structure of complex - exposed loop of enzyme binds in minor groove of DNA which mimics nicked DNA

Protein-Nucleic Acid Interactions Non-specific Interactions Exonucleases & endonucleases DNase I - forces (VDW contacts, H-bonds, salt bridges) Little seq-specificity

Protein-Nucleic Acid Interactions Non-specific Interactions Polymerases DNA-dependent DNA polymerase: E.Coli DNA pol I and III Pol III - chief replicative  subunit Fig 9.1 Pol 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 Interactions Polymerases DNA-dependent DNA polymerase: E.Coli DNA pol I and III Pol I - divalent cations important, may bind to catalytic AAs Cation 1 - positions OH- to attack phosphorus and generate pentacovalent transition state Cation 2 - stabilizes leaving group

Protein-Nucleic Acid Interactions Non-specific Interactions Polymerases RNA-dependent DNA polymerase: HIV Reverse transcriptase (RT) Uses DNA or RNA as template, no 3’-5’ proofreading P51/p66 heterodimer - has 5’-3’ pol site, RNase H site, Lys tRNA binding site which primes DNA synthesis

Protein-Nucleic Acid Interactions Non-specific Interactions Polymerases DNA bound to RT, protein interacts with phosphate backbone Conserved acidic residues (Asp110, Asp185, Asp186)