DNA transcription

DNA background

Must replicate faithfully

Coding capacity yo generate proteins

Transmitted parent to daughter cell

Composed of

Carbon

Hydrogen

phosphorous

Nitrogen

Oxygen

Nucleotides

phosphate +sugar+base

5' phosphate

3' hydroxyl

polymer

dAMP

dGMP

dCMP

dTMP

link can only be made between 5' of one and 3' of another

end groups determne directionality

3D structure

x-ray crystallography

model building

precipitated from solution by addition of ethanol

2-stranded helical structure with sugar-phospahte backbone

Chargaff's rules

A=T

G=C

bases exist in keto form, not enol form

Strands anti-parallel

Replication

Parent strand acts as template

3 models

  1. semi-conservative replication
  1. Conservative replication
  1. Dispersive replication

Each strand acts as template on which other strand is synthesised

Meselson-stahl experiment

Extracts DNA to investigate banding position

N-15 bands near bottom

N-14 bands near top

template-directed

Requirements

Must allow DNA double helix to unwind

Other enzymes must allow template-driven copying

Others mediate error correction

Key enzymes

Helicase

Unwinds Dna

Topoisomerase

Positive supercoiling

Polymerase and primase

DNA replication

Dealing with overwinding

Assay

Total protein and cell extract

Template DNA

Substrates for DNA synthesis

Nucleotide triphosphates

dATP

dTTP

dGTP

dCTP

Arthur Kornberg

Rules

1 DNAp can't start new DNA strand on its own

  1. 5' to 3' direction

Replication fork

Junction of unwinding molecules

Each replicated DNA molecule has old and new strand

Leadng strand

Continuous

Lagging strand

Discontinuous

Okazaki fragments

DNAP1

5' to 3' DNA polymerizing

3' to 5' exonuclease

Backwards

Forward synthesis

5' to 3' exonuclease

Forward, primer removal

Proofreading

DNAP III

principal

v fast

Starting primer

Initiation sensitive to rifampicin

inhibitor of RNAP

No longer inhibits after it gets started

Thus primer needed to start

3H-Uracil

Get shorter when treated with alkali

joining up

dnap III adds DNA nucleotides to 3' end of RNA primer

  1. DNAP III approaches next primer
  1. DNAPI takes over, replaces RNA primer with new DNA

click to edit

Nick translation

  1. DNA ligase seals nicks

Polymerase chain reaction

oligonucleotides

21-29 base

Pair used to prime amplification of target DNA sequence in cyclical manner

DNA sequencing

Sequencing by synthesis

Fred Sanger

Development of di-deoxynucleotides

Terminate chain synthesis

Separates newly synthesised strands by size

Fluorescently labelled with fluorophore

Role of RNA

synthesised in nucleus

Migrates to cytoplasm where proteins are synthesized

precedes new viral particle formation

Structure

Polymer with ribose-phosphate backbone

AG, UC

Sugar is ribose

single stranded polymer

Prokaryotic transcription

Promoters

DNA sequences which guide RNAP to start transcription

Terminators

initiates, elongates, terminates

DNA ccmpared to RNA

nucleus within double membrane

DNA is wrapped around histones to give chromatin

promoters have many control sites for transcriptions for TFs

RNAP has 12 protein subunits, associating with many different regulatory proteins

Opening and closing chromatin controls transcription in eukaryotes

access to core promoter regulated by TFs and chromatin stat