BIMM 100 MT 1

Hershey-Chase Experiments (1952)

Proved deoxyribonucleic acid (DNA), rather than protein, is the genetic material.

Run by infecting E. coli bacteria with phages containing radioactively labeled DNA (³²P) or protein (³⁵S), then agitating them in a blender to detach the empty viral coats. Finally, the mixture was spun in a centrifuge to separate the heavy bacterial cells from the viral debris, revealing that only the labeled DNA had entered the bacteria.

Nucleotide Requirements

1. Phosphate Group

2. (deoxy) Ribose / sugar pentose

3. Base

Purines

Bases with two rings: Adenine (A) and Guanine (G)

Pyrimidines

Bases with one ring: Uracil (U), Thymine (T), Cytosine (C)

Adenine

Forms two hydrogen bonds

Guanine

Forms three hydrogen bonds

Thymine

Forms two hydrogen bonds

Cytosine

Forms three hydrogen bonds

Uracil

Found only in RNA

Chargaff’s Rules (1947)

A = T, G = C

T_m

Melting temperature, the temperature at which half the bases of a dsDNA become denatured. Higher T_m corresponds to higher G-C content since they have 3 hydrogen bonds, making them more resilient to denaturing.

Gene

The entire nucleic acid sequence that is necessary for the synthesis of a functional product.

Includes:

• coding region

• transcribed non-coding regions (99% of the human genome)

  • UTRs (untranslated regions)

  • introns

• transcriptional control regions

• splice sites

• polyA sites

Solitary Genes

• found only once in the genome

• core, irreplaceable housekeeping functions

Duplicated Genes

Multiple genes of close but not identical sequences.

• often within 5-50kb of one another

• gene families encode homologous (closely related) proteins

• source of evolutionary novelty and specialization

How are genes duplicated?

Pairing between repeat sequences (L1) allows for unequal recombination

Tandemly Repeated Genes

Encode identical or nearly identical gene products.

(E.g rRNAs, tRNAs, histone coding genes)

DNA Transposon

Cut & paste mechanism into different position in the genome

Retrotransposon (RNA)

Copy & paste mechanism into different position in the genome.

DNA Transposons

Transposase

1. Blunt cut (cuts through both stands) at specific sites in donor DNA

2. Staggered cut (cuts through one strand) in target DNA

3. Ligation of donor to target

LTR (Long Terminal Repeat) Retrotransposons

Reverse Transcriptase (RT) - copies RNA into DNA

Integrase - inserts the NDA into the Genome

Non-LTR Retrotransposons

• LINEs

  • Long Interspersed Elements

  • encode proteins that enable mobility

  • 21% of total human DNA

• SINEs

  • Short Interspersed Elements

  • rely on LINE protein for retrotransposition

  • 13% of total human DNA

Transposons and Evolution

1. Generation of gene families - duplications

2. Creation of new genes - exon shuffling

3. Change regulatory regions that control gene expression

Chromatin

Disorganized chain of nucleosomes packed together at different concentration densities.

1. DNA (wraps around nucleosomes)

2. Histones (major ones: H1, H2A, H2B, H3, H4)

3. Other proteins (scaffold, replication, and transcription factors)

Histone Tails

The termini of the histone proteins; beginning and end of proteins. Modification (eg. methylation & acetylation) controls chromatin condensation and function.

Acetylation

Facilitated by Histone Acetyltransferase (HAT). Acetylates lysine 9 on histone 3 (H3K9) and removes the positive charge → looser

Methylation

Facilitated by Histone Methyltransferase (HMT). Methylates lysine 9 on histone 3 → tighter

Trimethylation of lysine 9 (H3K9Me₃) allows HP1 to bind

Euchromatin

Loose chromatin that enables transcription/gene expression.

Heterochromatin

Tight chromatin that prevents transcription/gene expression.

Heterochromatin Protein 1 (HP1)

Promotes heterochromatin formation; binds to trimethylated lysine 9 on H3 and pulls other HP1 (oligomerization) & binds to H3K9 HMT → pulls histone together

Boundary Element (Insulator)

sequence of DNA that prevents spreading of a chromatin state, for example, by being bound to non-histone proteins

DNase I Protection Assay

Used to determine the state of chromatin of a gene

1. Isolate nuclei

2. DNase I treat sample

3. Purify DNA (remove DNase) and digest with Bam HI

4. Southern Blot analysis with labeled probe designed to hybridize to part of gene

Southern Hybridization/Southern Blot (DNA)

1. Digest DNA with restriction enzyme to cut into smaller pieces

2. Separate using gel electrophoresis

3. Transfer from gel to nitrocellulose

4. Hybridize with labeled (eg. fluorescence or radiation) DNA or RNA probe complementary to target sequence

5. Detect (eg. via autoradiogram)

Karyotype

The number, size, and shape of metaphase chromosomes in a species

Elements for Chromosomal Replication and Inheritence

1. Replication origins (ORI) - replicate DNA

2. Centromeres (CEN) - proper segregation of daughter chromosomes

3. Telomeres (TEL) - protect ends

DNA Replication Requirements

1. Template

2. Primer

3. dNTP’s

4. Proteins

Topoisomerase

Relieves the stress of DNA unwinding done by Helicase

Lagging Strand

• multiple RNA primers

• extended 5’ to 3’ by DNA pol δ

• RNA degraded

• DNA pol fills gap

• ligation

Leading Strand

• 1 RNA primer

• extended 5’ to 3’ by DNA pol ε

DNA Replication Proteins Order

1. Large T antigen - helicase

2. RPA (Replication Protein A) - binds unwound parent DNA and keeps it unwound

3. Topoisomerase - removes stress in front of helicase

4. Primase (RNA Pol), DNA Pol α - adds dNTPs to both leading and lagging strands

5. RFC (Replication Factor C) & PCNA (Proliferating Cell Nuclear Ant.) clamp on Pol; DNA Pol δ - synthesizes lagging strand

6. RNase H + FEN1 - remove primers

7. DNA Ligase - connect DNA fragments

8. RFC/PCNA - clamps on DNA Pol ε for synthesis of leading strand

High Fidelity DNA Replication Proteins

DNA Pol δ & DNA Pol ε

Low Fidelity DNA Replication Proteins

Primase & DNA Pol α

Telomerase

• RNA serves as template

• Reverse transcriptase protein carries out reverse transcription

Polymerase Chain Reaction (PCR)

Amplifies a specific DNA sequence from a DNA sample. Requires:

1. Template DNA

2. DNA primers

   • DNA instead of RNA to prevent need for removal → no end-replication problem

3. dNTPs

4. Polymerase

When used to detect a sequence of interest after DNase I Protection Assay, the presence of a PCR product indicates heterochromatin, while the absence indicates euchromatin

PCR Cycle

1. Denaturation of DNA & annealing of primers

2. Elongation of primers

3. Repeat in next cycle

DNA Fingerprinting

• Based on Simple Sequence Repeat (SSR) DNA

  • Sequences of SSR’s are highly conserved but the number of repeats vary

• 1 band = 1 set of primers

Proofreading

A type of DNA repair done by DNA Pol δ and ε, which have 3’ → 5’ exonuclease activity. Steps:

1. Pol pauses

2. Pol removes nucleotide

3. Pol resumes replication

Mismatch Excision Repair

Corrects single stranded replication errors.

1. Incorrect base in daughter strand is recognized

2. The nucleotide with the incorrect base and surrounding nts are removed

3. Gap is repaired by DNA Pol δ/ε and ligase

Base Excision Repair

Replaces a chemically changed base (eg. deamination of cytosine → uracil → thymine) in ssDNA

1. Remove incorrect base (eg. T)

2. Endonucleases remove deoxyribose phosphate

3. Gap is filled with correct base by DNA Pol β and ligase

Nucleotide Excision Repair

Fixes distorted ssDNA (eg. formation of Thymine dimers)

1. Distortion is recognized

2. Helix opened

3. Endonucleases remove damaged and surrounding nucleotides

4. Gap is repaired by DNA Pol δ/ε and ligase

Homologous Recombination

An error-free type of dsDNA repair. Undamaged homologous chromosome serves as template.

1. dsDNA break is recognized and exonucleases remove nucleotides, leaving 3’ end ssDNA overhangs

2. ssDNA searches for homologous sequence in sister chromatid

3. Synthesis by DNA Pol using 3’ end of broken DNA as a primer and sister DNA as template

4. Ligation

5. Resolution to 2 intact dsDNAs, Holliday structure formed

End-Joining

An error-prone type of dsDNA repair. Two separated ends rejoined or different broken ends joined (translocation).

1. Recognition of broken ends

2. Exonucleases remove overhang nucleotides

3. Ligation

Translocations

Broken ends from different chromosomes are end-joined

RNA Transcription Requirements

1. RNA Polymerase/proteins

2. Template

3. rNTPs

NO PRIMER NEEDED

Transcription Stages

Initiation:

1. Polymerase binds to promoter sequence in duplex DNA - “Closed complex”

2. Polymerase melts duplex DNA near transcription start site, forming a transcription bubble - “Open complex”

3. Polymerase catalyzes phosphodiester linkage of two initial rNTPs

Elongation:

4. Polymerase advances 3’ → 5’ downstream (+) the template strand, melting duplex DNA and adding rNTPS to growing RNA

   • note that synthesis is stll 5’ → 3’

Termination:

5. At transcription stop site, polymerase releases completed RNA and dissociates from DNA

Prokaryotic Operon (Gene Expression) vs Eukaryotic Gene Expression

• 1 Promoter vs 5 Promoters

• 1 Transcription stop site vs 5 transcription stop sites

• 1 mRNA vs 5 mRNA

• 5 Proteins produced (polycistronic) vs 5 proteins produced

Northern Blot (RNA)

1. Isolate total RNA

2. Denature and separate RNA by electrophoresis (no restriction enzymes needed, only in dsDNA)

3. Transfer to nitrocellulose and fix

4. Hybridize to probe (must be complementary)

5. Detect (RNA or DNA probe)

Sigma (σ) Factors

1. Requires for RNA Pol to initiate transcription

2. Bind RNA Pol and promoter sequences in DNA

3. Assist RNA Pol in separating DNA strands

Lac Operon

Encodes 3 proteins needed for lactose metabolism - produces glucose and galactose

Repressed State (No Lactose):

1. Lac repressor protein complex binds the lac operator DNA

2. RNA Pol-σ⁷⁰ cannot bind promoter

Low Expression (Lactose + Glucose):

1. Lactose molecules bind lac repressor causing it to dissociate from the operator

2. RNA Pol-σ⁷⁰ can bind promoter and initiate transcription

   • Only binds to weak promoter (doesn’t match consensus sequence)

Activated State (Lactose Only):

1. Lactose molecules bind lac repressor causing it to dissociate from the operator

2. Low glucose = hi cAMP

   • cAMP binds CAP proteins, allowing them to bind the CAP site

3. CAP + cAMP bind to CAP site, stimulates RNA Pol-σ⁷⁰ to increase transcription initiation

Eukaryotic Transcriptional Control

• Activators (decondenses heterochromatin to form euchromatin)

  • Bind DNA

  • Recruit co-activators

    • HATs (histonemethyltransferase)

    • Mediator

• Repressors (condenses euchromatin to form heterochromatin)

  • Bind DNA

  • Recruit co-repressors

    • Histone deacetylases

    • HMTs (histonemethyltransferase)

Transcription Regulation

Promoter - sequence of DNA for RNA polymerase to bind to for transcription

Transcription Factors (TF) - proteins that regulate transcription

RNA Polymerase II Promoters

General Promoter Elements:

1. TATA boxes

   • positions RNA Pol II for transcription

2. Initiator

   • at start site

   • +1

3. CpG Islands

   • usually 100-1000nt upstream of start site

Divergent Transcription

• Transcripts made in both directions

• Usually from CpG promoters

• Wrong direction transcripts usually terminate and are unusable

General Transcription Factors

1. Sequential binding of general transcription factors (TFIIs + Pol II)

   • position polymerase

   • unwind DNA

Assembly of Pol II Pre-Initiation Complex (PIC)

1. TFIID

   • TBP = TATA Binding Protein

   • TAFs = TBP Associated Factors

2. TFIIA, TFIIB, TFIIF, Pol II, TFIIE, TFIIH (helicase)

   • unwind template

   • start synthesis

   • phosphorylation of Pol II CTD (Carboxyl-Terminal Domain, 7 amino acid repeats)

Systematic Mutagenesis

Determines which promoter sequences are regulate/allow transcription

1. Create reporter genes

   • introduce mutations (changes in nucleotide sequence) at specific positions in control region sequence

2. Put reporter in expression system

3. Assay reporter expression

DNase I Footprinting

In vitro; Reveals whether or not a protein binds to a DNA sequence and where

1. ³²P-label dsDNA at one end

2. Incubate DNA with protein sample

3. Add DNase I (limiting amount)

4. Isolate DNA

5. Denature and separate by electrophoresis

6. Detect labeled DNA fragments

   • No cuts by DNase where the protein is sitting

Electrophoretic Mobility Shift Assay (EMSA)

In vitro - Shows binding affinity but not precise location

1. Incubate ³²P-labeled dsDNA probe with protein

2. Electrophorese under non-denaturing conditions

ChIP (Chromatin Immunoprecipitation)

Used to reveal DNA-Protein interactions

1. Cross-link, then isolate chromatin

2. Break chromatin into pieces

3. Use specific antibody to select chromatin pieces containing protein of interest

4. Release DNA

5. Use PCR to determine if a specific gene/DNA sequence was associated with the protein of interest

Nuclear Hormone Receptors

Type of TF

1. Bind hormone (ligand)

2. Bind DNA as dimers

3. Regulate txn

Homodimeric Nuclear Hormone Receptors

1. - Ligand = cytoplasmic → No txn

2. + Ligand = nuclear translocation → Activation of txn

Heterodimeric Nuclear Hormone Receptors

1. Repress txn in absence of ligand

2. Activate txn upon binding ligand