BIOL 308 - Lectures 2-5

Functions of DNA?

• 3 main functions

1. store information

2. replicates faithfully (preservation of information)

3. has ability to mutate (variability of information)

Central Dogma of Moleular Biology

DNA → RNA → Protein

• DNA undergoes transcription → RNA undergoes translation → Protein

Gene

• entire DNA sequence necessary for production of functional protein or RNA

  • coding sequences for proteins and RNAs;

  • regulatory sequences act as signals or binding sites

Initiation of Gene Expression

• transfer of information

• in replication of DNA there is a template strand = antisense strand and an RNA like strand = sense/coding strand

DNA is double helix

• DNA strands have → polarity

• they are also complementary and antiparallel

2. Replicates faithfully

• two strand of a parallel DNA separate and each serves as a template for synthesis of a new daughter strand by complementary base pairing

• one strand predicts the sequence of the other strand → replication is semiconservative

Meselson and Stahl: thinking/planning

• Tried different models: Semiconservative, conservative, and dispersive models

• used E. Coli with heavy and light N to see which ones would appear after replication

Meselson and Stahl: Outcome

• DNA replication is semiconservative

  • two strands of a parental DNA separate and each serves as a template for synthesis of a new daughter strand by complementary base pairing

• Outcome: one strand predicts the sequence of the other strand - information is preserved

3. has ability to mutate

• mutations in coding sequences → possible alteration in protein product

• concept of collinearity of genes and proteins

• mutations could happen in regulatory sequences

• Importance of mutations → formation of new alleles

  • altered product (= protein or RNA)

  • no product (knock out)

  • altered regulation of product expression (if mutation in regulatory sequence) → + selection = EVOLUTION

Overview of nucleic acid structures

• Bases:

  • purines (A&G) and pyrimidines (C, T, U)

• Sugars:

  • 2-deoxyribose (DNA) → has a 2- H and 3’ OH

  • ribose (RNA) → has a 2- OH and 3’ OH

• Phosphate → PO4

Makeup of a Polynucleotide chain

• the H of the 3’ OH binds to the HO of a phosphate on another nucleotide forming a phosphodiester linkage

• chains are read in the 5’ to 3’ direction, both ends running antiparallel to one another

Chargaff’s rule

• #purines = #pyrimidines

• #A=#T and #G=#C

Major and Minor Grooves

• binding sites for different (regulatory) factors

• each factor recognizes specific nucleotide sequence on DNA

• each nucleotide sequence “exposes” specific - unique - distribution of acceptors and donors

Forces that help form DNA double helix

1. Rigid phosphate backbone - overall negative charge to the molecule

2. Stacking interactions - Van der Waals interactions between bases (weak, but many)

3. Hydrophobic interactions - highly negative phosphate backbone “outside” vs. nonpolar "(hydrophobic) bases “inside”

4. Ionic Interactions - salts (+ve ions) stabilize phosphate backbone (DNA shielding)

5. Hydrogen Bonding is responsible for complementary base pairing but is not the most energetically significant component

Alternative Forms of DNA: A-DNA

• Orientation: right-handed orientation

• major grooves: deep and narrow

• minor grooves: shallow and broad (superficial),

• conditions: low humidity (75%) and high salt

Alternative Forms of DNA: B-DNA

• Orientation: right-handed orientation

• major grooves: moderate depth and wide

• minor grooves: moderate depth and narrow

• turning: every 10.5 bases

• conditions: high humidity (95%) and low salt

Alternative Forms of DNA: Z-DNA

• orientation: left-handed orientation

• major grooves:

• very shallow, virtually nonexistent “single groove”

• minor grooves: very deep and narrow

• conditions: with very high MgCl2, NaCl or ethanol. In the presence of methylated cytosine: high humidity and low salt

Unusual Forms of DNA

• Triple helix DNA:

  • formed when purines make up one strand and pyrimidines the other, then a third strand can be accommodated

  • in a test tube, but also likely in vivo during DNA recombination or repair

  • gene therapy possibilities

Chromosomal DNA is a dynamic structure

• localized structural polymorphisms

  • constant

  • DNA sequence

  • local environment

• allows for recognition of DNA

  • gene expression

  • DNA repair

Denaturation of DNA

• two sequences that are not complementary will not hybridize

• two DNA molecules previously denatured by heating

• slow renaturation by cooling

• renatured DNA” 2 wild type molecules and two hybrid molecules

Factors that Denature DNA

• heat

• low ionic strength promotes repulsion between negative phosphate back-bones (low salt)

• high pH: “stripping” of H+ shared between electronegative centers (NaOH)

• agents that influence H-bonds

  • competition: have functional groups that can form H-bonds with the electronegative centers (NH2- and O=; urea, formamide)

  • covalent modifications: modify electronegative centers and block the formation of H-bonds (formaldehyde, glyoxal)

• agents that enhance the solubility of hydrophobic substances (organic solvents, temperature, pH)

Monitoring DNA Denaturation

• progress of denaturation can be monitored by examining the properties of the molecule that change when the strands separate

  • viscosity - rarely used = difficult

  • absorbance - commonly used

How does absorption spectrophotometry work?

• Tm: melting temperature (temp at which 50% of the DNA is denatured

• absorbance changes depending on the stacking of purines and pyrimidines:

  • in double stranded DNA that bases are stacked and absorbance if lower (hypochromic)

  • in denatured single stranded DNA the bases are unstacked and absorbance increases (hyperchromic)

Denaturation and GC content

• Tm is a function of the GC content

• more GC: higher Tm needed

• AT regions separate first during denaturation

• the Tm of DNA increases by 0.4°C with every 1% increase in G-C content under normal condition

• higher salt = higher Tm

Renaturation

• is the recombination of two complementary single stranded DNA

• dependent on: DNA concentration - complementary single strands must “fine each other” (number of copies)

• Salt concentration - ionic conditions - mask repulsion forces of phosphate backbone

• temperature: 20-25°C below Tm

• Time (reaction time)

• Size of the DNA fragment (length)

• Complexity - simple sequences re-nature faster than complex sequences

• these properties are used to analyze and classify DNA

CoT analysis

• rate of renaturation = measure of complexity of DNA/genome

• re-association kinetics: speed at which a single strand sequence is able to find a complementary sequence and base pair with it

• expect: increase in genome size = increase in complexity

• simple sequences re-nature more quickly than complex sequences

• Co= starting concentration

• t = rxn time (in sec)

Cot analysis conditions

• units of complexity are measured in terms of nucleotides

• if a genome is all unique (nonrepeating_ in a sequence then: complexity = # of nucleotides

• if a genome contains unique sequences and some repetitive sequences then: complexity = # of unique nucleotides + total # of nucleotides from one copy to each repetitive sequence

• if two DNA sequences do not have repetitive sequences and have similar C-G contents, their sizes are proportional to their Cot ½

Complexity examples

• DNA composed of the repeating copolymer dAT (ATATATATAT) has a complexity of 2

• DNA composed of the repeating tetrameric sequence (ATGC) n has a complexity of 4

• A DNA composed of 10 5 non-repeating nucleotide pairs in length has a complexity of 10⁵

• A DNA composed of 10 5 non-repeating nucleotide pairs, plus 100 copies of dAT, 50 copies of (ATGC) in length has a complexity of 10⁵ + 2 + 4

How is a Cot analysis carried out?

1. take a control DNA which is known and 100% complementary and unknown DNA

2. Shear them into small pieces (approx. 200bp)

3. Denature with heat

4. allowed to cool slowly (re-anneal)

5. Sub-samples removed: ds & ss DNA measured (abs at 260nm measured over time decrease during renaturation)

6. Data points plotted as a proportion of ssDNA (or %dsDNA) out of the total DNA

E. coli Genome vs. Calf Genome

• e. coli: no repetitive sequences - genome is one unique sequence

  • difficult for sequences to find complementary sequences

  • once they are found - fast re-association

• calf: lots of highly repetitive sequences - fast re-association

  • some moderately repetitive sequences slower re-association at the beginning

  • slowest (those unique sequences are comparable to E. coli genome)

Highly Repetitive

often short sequences, but there are many of them, they are able to find each other very easily → fast renaturation.

Moderately repetitive

moderate # (10-100), find each other with little difficulties, need little more time → middle renaturation. Some of them lack coding function. Some of them - code for different gene families: globin genes, immunoglobulin genes, genes for tRNAs and rRNAs etc.

Unique repitition

one to few copies, have lots of difficulties to find each other during renaturation → slow renaturation → mostly protein coding sequences

Reassociation is inversely proportional to the genome (DNA) size

• the DNA sources are:

  • synthetic DNA duplex of poly A and poly U polynucleotide chains;

  • MS-2 DNA (bacteriophage); T4 DNA (more complex bacteriophages then MS-2)

  • E. coli DNA

• if a genome does not contain repetitive sequences, the complexity of the genome (expressed in # of nucleotides) is the same as the genome size

Determining Genome Size by Cot Analysis

• calculate the ratio between the rate of renaturation (Cot ½) of known unique sequence DNA and that of the unique fraction of unknown test sample

1. Cot ½ (known)/ Cot ½ (test) = size of genome (=4 × 10⁶bp)/size of unique fraction of test genome

2. 5/1000 = 4 × 10⁶ bp/x = 8 × 10⁸ = size of the unique part of the unknown genome which represents about 60% of the whole

3. x/100 = 8 × 10⁸ bp/60; x = approx size of total unknown genome = 1.3 × 10⁹ bp

Cot analysis revived: aids genome sequencing

• unique, protein coding sequences could be “purifies” from the mix

• faster sequencing

• cheaper sequencing

Is complexity of genome correlated with the biological complexity of the organism?

• N = haploid chromosome #

• C = DNA/haploid cell

  • C varies greatly; general increase from prokaryotes to eukaryotes; large differences within eukaryotes

• C value paradox

  • no correlation between the amount of DNA (size of genome) and the apparent complexity of organisms

Circular DNA

• is composed of 2 strands of DNA that form a closed structure without free ends = “double circle”

• prokaryotic genomic DNAs, plasmids and many viral DNAs are circular

• chloroplast and mitochondria also have circular genomes = endosymbiotic theory

Denaturation of circular DNA

• circular DNA can also be denatured like linear DNA

• however, two strands cannot unwind and separate like linear DNA

  • in vivo, nicking occurs naturally during DNA rep.

  • can be induced experimentally by using an enzyme

Both circular and linear DNA:

• primary structure of DNA: sugar-phosphate “chain” with purine and pyrimidine bases as side chain(s)

• secondary structure of DNA double helical structure (H bonds between A-T and G-C; stacking interactions; phosphate backbone “outside"“)

• Tertiary/higher order structure: double stranded DNA (both circular and linear) make complexes with proteins - supercoil

Supercoiling

• “coiling of a coil”

• reduce stress on DNA by twisting/untwisting double helix

• Topological isomers - DNA differing only in their states of supercoiling

• important for packing of DNA - circular of linear

• DNA helix become topographically linearized (locally uncoiled) during replication and transcription

  • base pairing is interrupted

  • DNA molecules exhibits supercoiling

Positive and Negative Supercoils

• when a protein opens double-stranded DNA

  • in the front of the opening: DNA becomes overwound (more twists than normal) → creates a + supercoil, DNA is under greater torsional stress

  • behind the opening: DNA becomes underwound (fewer twists than normal) → creates - supercoils, DNA is more relaxed and tends to unwind;

Topology Numbers

• Twisting Numbers (T): measures how tightly the two DNA strands twist around each other

  • Right-handed DNA: +T, left-handed DNA: -T

• Writhing Number (W): measures supercoiling (how many times DNA helix crosses over itself)

  • Relaxed DNA: W = 0; -ve supercoils: W< 0; +ve supercoils: W> 0

• Linking Number (L): Total number of times one DNA strand wraps around the other

  • includes both normal and twisting and supercoiling: L = T + W

  • for closed circular DNA, L is constant unless DNA is cut

Topoisomerases

• are enzymes that recognize and regulate supercoiling and play an important role in replication and transcription

• Most cell DNA are negatively supercoiled

• negative supercoils store energy - energy of negative supercoils can be converted into unwinding of double helix

• DNA overwound: +ve supercoiling: reduce chance of DNA-protein interaction

• DNA underwound: -ve supercoiling: store energy that could help strand separation - unwinding favoured (for rep and transcription)

Prokaryotic

• + and - supercoils essential in prokaryotes - studies with mutants

• topoisomerase I: nicking-closing enzyme, makes transient cuts in one strand - relaxes negative supercoiling in one strand - relaxes (-) supercoiling in prokaryotes, changes L# in steps of 1

• topoisomerase II - relaxes + supercoiling (uses ATP). Makes double-stranded cut, pass a duplex DNA through it and re-seals the cut, changes L# in steps of 2

• Gyrase (one of bacterial Topo II): introduces - supercoils

• Reverse gyrase - in hyperthermophillic archaea, topo I generates + supercoils (requires ATP)

  • stabilizing the genome structure at high temperature (genetic knock-out experiments: reverse gyrase mutant is viable but shows significant growth defects at high temperature)

  • protecting the DNA strand breakage promoted by exposing DNA to high temperature

mRNA

• messenger RNA, specifies order of amino acids during protein synthesis

tRNA

• transfer RNA, during translation mRNA information is interpreted by tRNA

rRNA

• ribosomal RNA, combines with proteins aids tRNA in translation

Small RNAs

variety of regulatory functions

Riboenzymes

RNAs with enzymatic functions (in splicing, and peptide bond formation during protein synthesis)

RNA functions

• Noncoding RNAs: Regulatory

• Coding RNAs: code for proteins

• Noncoding RNAs: riboenzymes, transport