Lecture 8 Nucleic Acids

The DNA Duplex Can Be Reversibly Denatured (Melted)

Tm (transition midpoint) as a function of base composition

• Salt dependence is more dramatic

Hybridization

• DNA sequences can spontaneously re-anneal and form helices

• Basis for many of molecular biology techniques.– PCR, DNA

sequencing

PCR

•When a sample of DNA is too small to be sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies ("amplify") of it.

•PCR amplifies DNA by repetitive cycles of the following steps.

• 1. Denaturation2. Annealing ("priming")3. Synthesis ("extension" or "elongation")

PCR

Target regionTarget region

((aa) Consider double-stranded DNA containing) Consider double-stranded DNA containinga polynucleotide sequence (the target region)a polynucleotide sequence (the target region)that you wish to amplify.that you wish to amplify.

((bb) Heating the DNA to about 95 ) Heating the DNA to about 95 ℃℃ causes the causes the strands to separate. This is the denaturation strands to separate. This is the denaturation step.step.

((cc) Cooling the sample to ~60 ) Cooling the sample to ~60 ℃℃ causes one causes oneprimer oligonucleotide to bind to one strand andprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is thethe other primer to the other strand. This is theannealing step.annealing step.

PCR

((cc) Cooling the sample to ~60 ) Cooling the sample to ~60 ℃℃ causes one causes oneprimer oligonucleotide to bind to one strand andprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is thethe other primer to the other strand. This is theannealing step.annealing step.

((dd) In the presence of four DNA nucleotides and) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is the enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisextended in its 3' direction. This is the synthesisstep and is carried out at 72 step and is carried out at 72 ℃℃..

PCR

DNA Polymerase Mechanism

This completes one cycle of PCR. This completes one cycle of PCR.

((ee) The next cycle begins with the denaturation) The next cycle begins with the denaturationof the two DNA molecules shown. Both areof the two DNA molecules shown. Both arethen primed as before.then primed as before.

PCR

((ff) Elongation of the primed fragments completes) Elongation of the primed fragments completesthe second PCR cycle.the second PCR cycle.

PCR

The two contain only the target region andThe two contain only the target region andincrease disproportionately in subsequent cycles.increase disproportionately in subsequent cycles.

PCR

PCR results

CycleCycle Total DNAsTotal DNAs Contain only targetContain only target0 (start)0 (start) 11 0011 22 0022 44 0033 88 2244 1616 8855 3232 22221010 1,0241,024 1,0041,0042020 1,048,5661,048,566 1,048,5261,048,5263030 1,073,741,8241,073,741,824 1,073,741,7641,073,741,764

The Genetic Code• The genetic code is found in the sequence of nucleotides in

mRNA that is translated from the DNA• A codon is a triplet of bases along the mRNA that codes for a

particular amino acid• Each of the 20 amino acids needed to build a protein has at least 2

codons• There are also codons that signal the “start” and “end” of a

polypeptide chain• The amino acid sequence of a protein can be determined by

reading the triplets in the DNA sequence that are complementary to the codons of the mRNA, or directly from the mRNA sequence

• The entire DNA sequence of several organisms, including humans, have been determined, however,- only primary structure can be determined this way- doesn’t give tertiary structure or protein function

mRNA Codons and Associated Amino Acids

Reading the Genetic Code

• Suppose we want to determine the amino acids coded for in the following section of a mRNA

5’—CCU —AGC—GGA—CUU—3’

• According to the genetic code, the amino acids for these codons are:

CCU = Proline AGC = Serine GGA = Glycine CUU = Leucine

• The mRNA section codes for the amino acid sequence of Pro—Ser—Gly—Leu

Messenger RNAs• Contain protein coding information

– ATG start codon to UAA, UAG, UGA Stop Codon

– A cistron is the unit of RNA that encodes one polypeptide chain

– Prokaryotic mRNAs are poly-cistronic

– Eukaryotic mRNAs are mono-cistronic

mRNA coding patterns

Transfer tRNA

•There are 20 different tRNAs, one for each amino acid.

•A particular amino acid is attached to the tRNA by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the tRNA.

Transfer RNA

•Example—Phenylalanine transfer RNA

One of the mRNA codons for phenylalanine is:One of the mRNA codons for phenylalanine is:

UUCUUC5'5' 3'3'

AAGAAG3'3' 5'5'

The complementary sequence in tRNA is calledThe complementary sequence in tRNA is calledthe the anticodonanticodon..

Phenylalanine tRNA

OCCHCHOCCHCH22CC66HH55

NHNH33

OO

Anticodon

3'

5'

Each tRNA is single stranded with a CCA triplet at its 3' end.

++

5'5'

3'

5'

Ribosomal Peptidyl Transferase Activity

Note: the catalytic component of the ribosome’s peptidyl transferase activity is RNA; it’s an example of a catalytic RNA or ribozyme.

Other Nucleic Acid Structures

Non-Watson-Crick Base Pairing,e.g., Hoogsteen Base Pairing

Allow the formation of triple-stranded helices

Triple Helical DNA: H-DNAH-DNA structure can form when you have a homopurine stretch on a strand (so homopyrimidine stretch on the other strand).

H-DNA has been implicated in the regulation of several genes.

quadruplex

Self-Complementary Nucleic Acid Strands and Hairpins

Palindromic DNA Sequences:Potential to Form Cruciform Structures (Double Hairpins)

Palindromes and Restriction Endonucleases

Another reason palindromes are important:Type II restriction enzymes are site-specific endonucleases used in molecular biology research

(such as gene cloning) that recognize specific palindromic DNA sequences.

DNA cleavage products:

Sticky ends (e.g., Eco RI):5’-G-3’ 5’-AATTC-3’3’-CTTAA-5’ 3’-G-5’

Blunt ends (e.g., Sma I):5’-CCC-3’ 5’-GGG-3’3’-GGG-5’ 3’-CCC-5’

X-ray crystalstructure ofEco RI bound to DNA

RNA Helices are short, bulges, loops

RNA Secondary Structure Maps

tRNA-Phe Structure level 2

tRNA - the prototype structure

Protein-Nucleic Acids Interaction

• Perspective

• Non-specific interactions

• Specific interactions

What functions that DNA-protein interactions are involved in?

DNA replication, DNA repair, DNA recombination, transcription etc.

Two effective techniques: X-ray crystallography

and NMR spectroscopy (<25 kDa).

Both are equally valid but neither is sufficient without detailed kinetic, thermodynamic, and site-directed mutagenesis studies.

One of the function: The need for packaging

The fundamental building block of chromatin in eukaryotes is the nucleosome, a protein-DNA complex.

The nucleosome core particle consists of 146 bp of DNA and eight small, highly basic histone proteins. The DNA wraps around the histone octomer to form a negative supercoil.

Bacteria also use small basic proteins to package DNA, such as the dimeric HU protein from E. coli.

The Nucleosome -DNA (146 bp) wrapped around octamer of core histone proteins (+ linker DNA = ~200 bp)

Nucleosome

Viruses are highly symmetric particles that can pack their nucleic acid genome efficiently inside the protein capsid.

Protein subunits containing many basic amino acids interact with the viral nucleic acid in a non-sequence-specific manner.

In the helical TMV, some sequence-specific contacts are involved

in directing assembly of the virus.

History of structure determination

Structure of DNA is regular: a list of the positions of the atoms in the double helix.

Proteins are much less regular, but it is more difficult to understand, e.g., repressors, polymerases.

Aaron Klug

"for his development of crystallographic electron microscopy and his structural elucidation of biologically important nucleic acid-protein complexes“ (1982)

Alex Rich Ss nucleic acid-binding protein

Roger Kornberg

"for his studies of the molecular basis of eukaryotic transcription“(2006)

The forces between proteins and nucleic acids

There are four major forces that occur when proteins and NA interact, but it is very difficult to ascribe precise changes in free energy of association to specific interactions between protein and NA.

• Electrostatic forces: salt bridges

• Dipolar forces: hydrogen bonds

• Entropic forces: the hydrophobic effect

• Dispersion forces: base stacking

Electrostatic forces: salt bridges

Electrostatic forces are long range, not very structure-specific, and contribute substantially to the overall free energy of association.

Salt bridges are electrostatic interactions between groups of opposite charge. They typically provide ~40 kJ/mol of stabilization per salt bridge.

In protein-NA complexes, they occur between the ionized phosphates of the NA and either the -ammonium group of lysine, the guanidinium group of arginine, or the protonated imidazole of histidine.

Hydrogen bonds are dipolar, short-range interactions that contribute little to the stability of the complex but much to its specificity.

Hydrogen bonds occur between the amino acid side chains, the backbone amides and carbonyls of the protein, and the bases and backbone sugar-phosphate oxygens of the NA.

Dipolar forces: hydrogen bonds

When protein-nucleic acid molecules are not complexed, all their exposed hydrogen bond donors and acceptors form hydrogen bonds to water.

Hydrogen bonds are very important in making sequence-specific protein-

nucleic acid interactions.

Entropic forces: the hydrophobic effect

Hydrophobic forces are short range, sensitive to structure, proportional to the size of the macromolecular interface.

Molecules of water leave the interface between a protein and a nucleic acid. Consequently, the surface of the protein and nucleic acid tend to be exactly complementary so that no unnecessary water molecules remain when the complex forms.

Dispersion forces: base stacking

Dispersion forces have the shortest range but are very important in base stacking in double-stranded nucleic acid and in the interaction of protein with ss nucleic acid.

Base stacking is caused by two kinds of interaction: the hydrophobic effect and dispersion forces.

van der Waals forces

For ds nucleic acid, dispersion forces are clearly important in maintaining the structure by base stacking.

For ss nucleic acid, they also help it to bind proteins because aromatic side chains can intercalate between the bases of a ss nucleic acid.

Geometric constraints imposed by the nucleic acid

All NA have repeating polyanionic backbones and so all proteins that bind them have strategically placed arginines and lysines that create an electrostatic field to neutralize the negative charge.

Contacts to the bases are called "direct readout"

because what contacts form depends directly on the sequence of the nucleic acid; distinguishing sequences by how the sequence affects the distortability or conformation of the nucleic acid is called "indirect readout".

Double-stranded B-DNA Simple model-building predicted two of the many ways in which

proteins interact with B-DNA by hydrogen-bonding: 1) an antiparallel -sheet interacting to the phosphate backbone in

the minor groove, 2) an -helix interacting with bases in the major groove.

Thus, to distinguish the cognate sequence from all others by direct readout alone, protein must form more than one hydrogen bond to some of the base-pairs in the major groove.

In specific protein B-DNA complexes, about 1/2 of the hydrogen bonds are to the bases and the other 1/2 to the phosphate backbone.

Single-stranded nucleic acidHydrophobic bases in ss nucleic acid are more exposed. Ss nucleic acid binding protein has more hydrophobic binding surface than ds nucleic acid binding protein .

The hydrophobic surface often contains aromatic groups which interact more effectively with the nucleic acid bases, and also an electrostatic field that neutralizes the charge of the phosphate backbone.

Possibly because the structure of RNA varies more than that of DNA, proteins seem to recognize RNAs in more ways than they recognize DNAs.

RNAs, even more than DNAs, may be recognized by indirect readout.

The kinetics of forming protein-nucleic acid complex

Two factors affect the rate of formation of all protein-nucleic acid complexes: random thermal diffusion and long-range, directional electrostatic attraction.

A "one-dimensonal random walk" can account for

the observed rate of genome sequence-specific protein-DNA complexes.

The protein first binds non-specifically to the DNA and then diffuses or jumps along the DNA until it finds the appropriate sequence.

Thus, all sequence-specific DNA binding proteins may bind DNA in two ways: one for tight, sequence-specific binding and the other for looser, non-sequence specific binding.

Protein-Nucleic Acids Interaction

• Perspective

• Non-specific interactions

• Specific interactions

Non-specific interactions

• Single-stranded nucleic acid binding proteins

• Non-sequence-specific nucleases

• Polynucleotide polymerases

• Topoisomerases

Single-stranded nucleic acid binding proteins

ssDNA is formed during replication and most organisms produce proteins to bind it. These proteins form an important but diverse group.

A model has been suggested in which lysines and arginines neutralize the DNA phosphate backbone and the bases stack against aromatic amino acid side chains.

Non-sequence-specific nucleases

All organisms must degrade nucleic acid during their life cycle. There is no one enzyme designed for this purpose, but rather a large number of enzymes with different specificities. These include exo- and endonucleases and enzymes specific for ss- and ds-nucleic acid and for base sequences.

e.g., RNase and DNase

RNase and DNase have different reaction mechanisms because RNase uses the ribose 2'-hydroxyl group, not present in DNA, to attack the 5'-phosphate ester linkage.

Ribonuclease A, barnase, and binase

RNase A is not sequence specific because it only interacts with the base at the active site;

all other contacts are electrostatic ones to the sugar-phosphate backbone.

Deoxyribonuclease I

DNase I cleaves different sequences with different rates because of sequence-dependent steric hindrance at the active site.

G-C tracts accommodate the catalytic loop better because they have wider minor grooves than A-T tracts.

Polynucleotide polymerases

There are four classes of template-directed polynucleotide polymerases: DNA- or RNA-dependent and DNA- or RNA-polymerizing.

All add nucleotides to the 3'-end of a growing polynucleotide chain but they differ widely in how accurately they replicate the nucleic acid (their fidelity) and how many nucleotides they add before dissociating (their processivity).

e.g., Pol I and RTase.

They have the same overall architecture for gripping a nucleic acid during polymerization. It is a domain that looks like a right hand, with palm, fingers, and thumb subdomains.

Part of the palm subdomain and the direction from which the nucleic acid approaches the active site is conserved in these two polymerases, their 3'-5' exonucleases, and RNase Hs may all use the same mechanism, which requires two divalent cations.

DNA-dependent DNA polymerases: E. coli DNA polymerase I (Pol I) and III

All cellular DNA-dependent DNA polymerases have a 3'-5' proof-reading exonuclease, require a primer to begin synthesis, and replicate their own nucleic acid the most faithfully.

The Klenow fragment of Pol I contains two widely-separated domains, one carrying the polymerase activity, and the other the 3'-5' proofreading exonuclease activity.

The DNA approaches the polymerase from exonuclease side and bends by 90o to enter the polymerase site.

The protein does not read the DNA sequence at all. Instead, when an incorrect base is added, the DNA strands separate and the daughter strand is therefore more likely to reach over to the exonuclease, which then removes the incorrect base.

RNA-dependent DNA polymerases: HIV-1 reverse transcriptase (RTase)

RTase is a unique heterodimer. Its two subunits have the same sequence yet fold differently. The p66 subunit folds into a polymerase domain and an RTase H domain.

RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA which is hybridized to DNA.

Topoisomerases

• Type I• Type II

Positive and Negative Supercoiling

positive supercoil =left-handed = overwound DNA

negative supercoil = right-handed = underwound DNA

L = T + W

• L or Lk = linking number (number of times one strand crosses the other)

• T = twist (number of helical turns; for B-DNA, T = # bp divided by ~10.5 bp/turn)

• W = writhe (number of supercoils)

(L0 = linking number of relaxed molecule = T, since W = 0 in relaxed molecule)

Type I Topoisomerases

•∆ L = ±1 per cycle•Cleaves a single strand•Passes broken single strand around the other, then rejoins strands•Does not require ATP•Relaxes supercoiled DNA

Ο Ο Ο Ο

Structure of a Type I Topoisomerase

Type II Topoisomerases

•∆ L = ±2 per cycle•Cleaves both strands•Passes unbroken part of duplex through double- strand break, then rejoins strands•Requires ATP•Relaxes supercoiled DNA•Some type II enzymes (like DNA gyrase) can add negative supercoils

Topological Interconversions Catalyzed by Type II Topoisomerase

Relaxation

Catenation and Decatenation

Knotting and Unknotting

X-Ray Crystal Structure of a Type II Topoisomerase

Protein-Nucleic Acids Interaction

• Perspective

• Non-specific interactions

• Specific interactions

Specific interactions

For a cell to function at all, proteins must distinguish one nucleic acid from another very accurately.

Proteins that bind specific nucleic acid sequences also bind non-specific ones.

The placement of an -helix in the major groove appears to be the most common way of recognizing a specific DNA sequence.

Other parts of the protein, which form hydrogen bonds and salt bridges to the DNA backbone, position the element on the DNA so that it can achieve recognition.

Direct readout of the DNA sequence, most often in the major groove, is an important part of sequence-specific binding but is by no means the only component.

The direct readout can involve hydrogen bonds (1) directly to side chains, (2) to the polypeptide backbone, or (3) through water molecules, or depend on hydrophobic interactions.

Indirect readout is also important: the correct DNA sequence may differ from canonical B-DNA in a way that increases the surface area buried, the electrostatic attraction, or the number of hydrogen bonds formed.

Oligomerization upon binding the correct sequence often increases affinity and specificity.

Transcriptional regulators: the helix-turn-helix motif

• The prokaryotic complexes• Eukaryotic complexes: the homeodomain

Exclusively eukaryotic transcriptional regulators: the zinc finger and leucine zipper

• The zinc finger proteins

The Cys2His2 zinc finger

The Cys4 nuclear receptors

The GAL4 zinc finger

• The leucine zipper

zinc finger proteins

• A zinc finger is a small protein structural motif

• Sequence-specific DNA-binding proteins

zinc finger proteins

• Individual zinc finger domains typically occur as tandem repeats with two, three, or more fingers comprising the DNA-binding domain of the protein.

• These tandem arrays can bind in the major groove of DNA.

• The α-helix of each domain can make sequence-specific contacts to DNA bases; residues from a single recognition helix can contact 4 or more bases.

-Sheet binding motifs

• The met repressor family

• The TFIID TATA-box binding protein

a general transcription factor

Restriction endonucleases: EcoRI and EcoRV

EcoRI and EcoRV have very different structures and interact with DNA differently: the former only in the major groove; the latter in both grooves.

However, both employ the same enzyme mechanism and catalytic residues and both achieve their high degree of sequence specificity similarly.

In the complex with cognate DNA, much of the free energy of binding has been used to drive the cognate DNA into an unfavorable conformation that places the scissile phosphodiester bond in the active site and completes the binding site for the essential Mg2+.