Fadool Exam 3

Griffith’s experiments

Heat-killed virulent bacteria could transform live non-virulent bacteria into the virulent strain. Some chemical substance is able to carry genetic information between organisms. Did not know what molecule was responsible.

Chargaff’s Findings

1. Across genome, [A] = [T] and [G] = [C]

2. In a single strand, %A ≈ %T and %G ≈ %C

Hershey & Chase Experiment

Radioactive tagging of phosphorus in DNA and sulfur in proteins. Virus injects DNA, not protein into cells to direct replication, so DNA must be molecule of inheritance, not protein.

A/T pair characteristics

2 hydrogen bonds = weaker association (lower T_m) than G/C pair

G/C pair characteristics

3 hydrogen bonds = stronger association (higher T_m) than A/T pair

4 Required Characteristics of Molecule of Inheritance

1. Storage of information

2. Ability for replication

3. Mechanism for expression

4. Allowance for variation (mutability)

mRNA

messenger RNA carries information from DNA for protein synthesis

tRNA

transfer RNA brings amino acids to ribosomes for protein synthesis

anticodon loop recognizes codon. 20 different enzymes for “charging” tRNA, 1 for each amino acid

small RNA

non-coding, bind DNA and RNA to regulate transcription and translation

CoT plot

Plots concentration of ssDNA over time as ssDNA anneals into dsDNA. More complex (less repetitive) genomes anneal much slower than less complex (more repetitive genomes). In eukaryotes, the curve is multi-phased. Highly repetitive genes anneal quickly (at beginning of plot) and unique, single copy genes anneal slowly (at the end of plot).

Taylor, Woods, and Hughes Experiment

Used auto-radiography to track nucleic acids following DNA synthesis. Determined that DNA replication is semiconservative.

Characteristics of prokaryotic genome

Single, circular chromosome

Single replication origin

Lack of nucleus and histones

Little to no RNA processing, but coding regions may overlap with different start sites (frameshifting)

Ligase

Mends breaks in DNA

Helicase

separates dsDNA

Gyrase

Removes twists in DNA (prevents kinking/knoting)

Clamp protein

Holds DNA template onto polymerase and maintains synthesis within discrete region

tau (τ) and gamma (γ) proteins 

Load clamp and maintain parallel synthesis of leading and lagging strands

Single Stranded Binding Proteins (SSBP)

Bind strands and prevent reannealing

OriC

Origin of replication for prokaryotic chromosome

Sequence specific

AT rich region (easier to break)

DnaA

Binds to OriC and wraps DNA to form complex in prokaryotes

Wrapping causes helix to pop open and bubble opens in AT-rich OriC region.

DnaB

Prokaryotic helicase

Breaks H bonds in dsDNA

Hexamer

DnaC

Chaperone protein in prokaryotes that aids in binding of Dna B to the open bubble at OriC

Topoisomerase II

Binds downstream of the bubble and introduces negative supercoiling

Breaks strands and flips strands to prevents knots in chromosome during replication

ATP-dependent

DnaG

Synthesizes RNA primer in prokaryotes

Undergoes polymerase switching with DNA polymerase 3 to begin replication of genome

DNA polymerase III

DNA-dep. DNA pol. in prokaryotes

Synthesizes strand in 5’-3’ direction; reads template 3’-5’

3’-5’ exonuclease activity gives proofreading activity

Lacks 5’-3’ exonuclease activity (cannot remove RNA primer)

DNA polymerase I

Polymerase switching with Pol III

5’-3’ exonuclease activity allows for removal of RNA primer

Nicks remain at ends of RNA primer (filled by ligase)

Tus protein

Blocks progress of helicase in prokaryotes (terminates replication)

Autonomous Replicating Sequences (ARS)

Origins of replication in eukaryotes.

100’s of ARS per chromosome

Higher # of ARS = Faster replication

Licensing Factors

Bind to 100-200 bp AT rich sequences during G1 to mark ARS

Degraded after DNA replication to prevent double replication

DNA pol α (alpha)

Eukaryotic primase

Both RNA and DNA synthesis activity

Synthesizes RNA primer then switches to DNA synthesis

Lacks 3’-5’ exonuclease (proofreading) activity

DNA pol ε (epsilon)

leading strand synthesis

3’-5’ exonuclease (proofreading) mechanism

Lacks 5’-3’ exonuclease activity (cannot remove RNA primer)

DNA pol δ (delta)

lagging strand synthesis

3’-5’ exonuclease (proofreading) mechanism

Lacks 5’-3’ exonuclease activity (cannot remove RNA primer)

DNA pol γ (gamma)

Replicates mitochondrial DNA

Encoded by nuclear gene

Proliferating Cell Nuclear Antigen (PCNA)

Eukaryotic clamp protein

Marks S phase cells in lab

euchromatin

loosely packed chromatin (less DNA modifications and lower histone occupancy)

Early replicating

heterochromatin

tightly packed chromatin (more DNA modifications and higher histone occupancy)

Late replicating

Chromosome Assembly Factor-1 (CAF-1)

Deposits newly synthesized histones onto replicated DNA. Old and new histones are randomly assorted between the template and new strands to conserve epigenetic marks.

Methyltransferase

Transfers methyl modifications to newly synthesized DNA strand

Werner Syndrome

Rapid, irreversible aging in children

Mutation in WRN helicase needed for telomere replication

Prokaryotic RNA polymerase

Synthesizes all necessary types of RNA

RNA Polymerase I

rRNA synthesis

RNA Polymerase II

Synthesizes pre-mRNA

RNA Polymerase III

small RNA (tRNA, snRNA, small rRNA)

RNA Polymerase IV and V

non-coding RNA and iRNA