DNA Structure

All nucleotides contain three components: 1. A nitrogen heterocyclic base 2. A pentose sugar 3. A phosphate residue Nucleic Acids DNA and RNA are nucleic acids, long, thread-like polymers made up of a linear array of monomers called nucleotides

Ribonucleotides have a 2’-OH Deoxyribonucleotides have a 2’-H Chemical Structure of DNA vs RNA

Structure of Nucleotide Bases Bases are classified as Pyrimidines or Purines

The nucleus contains the cell’s DNA (genome) RNA is synthesized in the nucleus and exported to the cytoplasm Nucleus Cytoplasm DNA RNA (mRNA) Proteins replication transcription translation

dA dG dTdC Deoxyribonucleotides found in DNA

Nucleotides are linked by phosphodiester bonds

Bases form a specific hydrogen bond pattern DNA is double stranded

The strands of DNA are antiparallel The strands are complimentary There are Hydrogen bond forces There are base stacking interactions There are 10 base pairs per turn Properties of a DNA double helix

DNA is a Double-Helix

RNase P M1 RNA Transcription of a DNA molecule results in a mRNA molecule that is single- stranded. RNA molecules do not have a regular structure like DNA. Structures of RNA molecules are complex and unique. RNA molecules can base pair with complementary DNA or RNA sequences. G pairs with C, A pairs with U, and G pairs with U. bulge internal loop hairpin

Electrostatic : Salt bridges Dipolar : Hydrogen bonds Entropic : The hydrophobic effect Dispersion : base stacking Forces between proteins and DNA

Water Metal ions Small organic molecules Drugs Carcinogens Antibiotics Proteins Major Groove Minor Groove Nucleic Acids interact reversibly with:

Groove Interactions in DNA Figure 7.4

The majority of the interactions between proteins and DNA are hydrogen bonds with functional groups in the major groove of the double-stranded DNA molecule. Each DNA binding protein recognizes specific sequences in the DNA. Hydrogen bonding with N 6 and N 7 of Adenine, O 6 and N 7 of Guanine, O 4 of Thymine, and N 4 of Cytosine is possible. Binding to nucleotides in the major groove

Electrostatic : Salt bridges Dipolar : Hydrogen bonds Entropic : The hydrophobic effect Dispersion : base stacking Forces between proteins and DNA

Electrostatic : Salt bridges Interaction between groups of opposite charge Occur between the ionized phosphates of the nucleic acid and either the  -amino group of lysine, the guanidinium group of arginine, or the protonated imidazole of histidine. Forces between proteins and DNA

Dipolar : Hydrogen bonds  -  +  -  + X – H Y – R X and Y are nitrogen and oxygen in biological systems Positioning of hydrogen bond donors (X) and acceptors (Y) is optimized between protein and DNA. Forces between proteins and DNA

DNA binding proteins contain amino acids that hydrogen bond to functional groups in the major groove of DNA

Entropic : The hydrophobic effect A complementary surface formed between a protein and a nucleic acid will release ordered water molecules at the surface of the protein or nucleic acid. The formerly ordered water molecules become part of the disordered bulk water, thus stabilizing the interaction through an increase in the entropy of the system. Consequently, the surfaces of the protein and nucleic acid tend to be exactly complementary, increasing the specificity of the interaction. Forces between proteins and DNA

Dispersion : base stacking Base stacking is dependent on the hydrophobic effect as well as dispersion (London) forces. Molecules with no net dipole can attract each other by a transient dipole-induced dipole effect. These forces are weak but do play a role in protein – nucleic acid interaction, specifically in base stacking. Forces between proteins and DNA

E. coli DNA polymerase III has a doughnut-shaped hole lined with positively-charged amino acid side chains that interact with the negatively-charged DNA strand

 -helix The catabolite activator protein (CAP) from E. coli uses alpha helices to interact with nucleotide bases in the major groove of the DNA helix. An amine-containing amino acid side chain often forms hydrogen bonds with major groove bases. A single amino acid may form hydrogen bonds with multiple, adjacent nucleotide bases, increasing sequence- specific interaction. Common amino acid: arginine or glutamine

Proteins often bind to specific sequences of DNA. Example: Restriction enzyme EcoRI binds to the DNA sequence 5’-GAATTC-3’ 3’-CTTAAG-5’ How do proteins find their target DNA sequence? 1. Randomly bind, dissociate, re-bind until they find their sequence? (Three-dimensional random walk) 2. Bind non-specifically and then slide along DNA until they find it? (One-dimensional walk) The kinetics of forming protein – DNA complexes

From the moment a new strand of DNA is synthesized to the moment it is degraded in a cell, there are proteins associated with it. Many of these proteins interact in a non-sequence specific manner. Many of the proteins are involved in packaging the DNA. Example: histone proteins that form the nucleosome Proteins that interact non-specifically with DNA interact with the negatively-charged ribose-phosphate backbone. Therefore, they have a high percentage of basic amino acid side chains such as lysine and arginine. Non-sequence specific protein – DNA interaction

Nucleosomes DNA in eukaryotic cells is packaged into nucleosomes, which contain proteins called histones. DNA wrapped around a histone core (side view)

For a cell to function, proteins must distinguish one nucleic acid sequence from another very accurately. Activators and repressors of transcription turn specific genes on and off. Common themes of protein - DNA interaction 1. Helix-turn-helix 2. Zinc finger 3. Leucine zipper Specific protein – DNA interactions

Often found in proteins that regulate gene transcription. The pair of  -helices stack to form a V shape with an angle of about 60º The first helix positions the second helix. The second helix binds to the DNA, projecting into the major groove and recognizing specific sequences. Shown is a helix-turn-helix motif from a homeodomain protein, A family of proteins that binds to eukaryotic DNA and regulate transcription of specific genes. Helix-turn-helix motif

Often found in proteins that regulate gene transcription. A zinc is coordinated to cysteine or histidine residues of the protein. An  -helix is inserted into the major groove and binds DNA. Shown is a zinc finger motif from a repressor protein from a phage, a bacterial virus zinc histidine cysteine Zinc finger motif

Often found in proteins that regulate gene transcription. A zinc is coordinated to cysteine or histidine residues of the protein. An  -helix is inserted into the major groove and binds DNA. Shown is a zinc finger motif from the glucocorticoid receptor, a protein that mediates hormone action. Two zincs are present. zinc cysteine Zinc finger motif

Often found in proteins that regulate gene transcription. Two alpha helices interact through interaction between hydrophobic leucine amino acid side chains on one side of the alpha helix. Shown is a leucine zipper protein Leucine zipper motif

Negative regulatory proteins bind to operator sequences in the DNA and prevent or weaken RNA polymerase binding

Most prokaryotic mRNA molecules are polycistronic, they encode multiple genes. These genes are usually involved in the same biochemical event. A single promoter controls the expression of these genes. This functional unit of DNA is called an operon.

A classical example of transcriptional regulation is lactose metabolism in E. coli. Proteins required for lactose metabolism in E. coli are encoded by the lac operon.

The E. coli lac operon lacI – encodes the Lac repressor protein lacZ – encodes  -galactosidase lacY – encodes galactose permease lacA – encodes transacetylase O 2 and O 3 are pseudooperators

The Lac repressor protein is thought to bind to the main operator and one of the pseudooperators, forming a loop in the DNA.

When lactose is present in high concentrations, the lactose metabolism gene products are needed in a cell. In the absence of lactose, the Lac repressor protein binds to the operator in the DNA, repressing transcription. The Lac repressor, however, binds to allolactose, a metabolite of lactose, inducing a conformational change that abolishes binding to the DNA operator sequence. Transcription is no longer repressed. - allolactose  transparent + allolactose  bold

DNA binding proteins contain amino acids that hydrogen bond to functional groups in the major groove of DNA.

DNA sequences recognized by regulatory proteins are often inverted repeats of a short DNA sequence. These repeats form a palindrome with two-fold symmetry about a central axis. Regulatory proteins are often dimeric. Each subunit binds to one strand of the DNA. 5’-TACGGTACTGTGCTCGAGCACTGCTGTACT-3’ 3’-ATGCCATGACACGAGCTCGTGACGACATGA-5’ central axis

The Lac repressor protein The Lac repressor is a tetramer of four identical protein subunits. There are DNA-binding domains on each subunit shown in blue. The allolactose binding domain (green) is connected to the DNA binding domain through linker helices (yellow). Tetramerization domains (red) form contacts between subunits.

The Lac repressor protein The Lac repressor is a tetramer of four identical protein subunits. There are DNA-binding domains on each subunit shown in blue. The allolactose binding domain (green) is connected to the DNA binding domain through linker helices (yellow). Tetramerization domains (red) form contacts between subunits.

The DNA binding domains of the Lac repressor contain a helix-turn-helix motif, a structure critical for the interaction of many proteins with DNA. helix turn helix

Lac repressor protein (lacI) Figure 8-21

Lac repressor bound to DNA Figure 8-22

Lac repressor bound to DNA Figure 8-23

Protein – DNA interactions Figures 8-16 and 8-17

Transcriptional elements of a eukaryotic structural gene Figure 9.1 page 151

Transcriptional Activation Figure 9.2 page 152

TATA box sequences

Structure of the TATA box binding protein Figure 9.4 page 155

Structure of TBP complexed with DNA Figure 9.5 page 156

DNA bound to TBP is bent Figure 9.6 page 156

Sequence specific interactions between TBP and DNA Figure 9.7 page 157

Transcription Factors Chapter 10

Helical wheels of DNA-binding domains of transcription factors Figure page 192

Regulatory proteins that function as dimers contain regions of amino acid sequence that mediate interaction between protein subunits. One common motif is the leucine zipper. Fig ’-TACGGTACTGTGCTCGAGCACTGCTGTACT-3’ 3’-ATGCCATGACACGAGCTCGTGACGACATGA-5’ central axis

The leucine residues of a leucine zipper provide hydrophobic interaction between alpha helices at regular intervals. Fig 28-14

Leucine Zipper Figure page 193

Heterodimerization of leucine zipper proteins Figure page 193

Transcription Factor GCN4 Figure page 194

DNA-binding domain of GCN4 Figure page 195

GCN4-DNA interactions Figure page 195