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Topic 7.

1 – DNA structure and replication


Hershey-Chase Experiment
- Confirmed DNA was the genetic
material of life
- Grew bacteriophage viruses in
two types of cultures
o Phosphorus-32 
radioactive DNA
o Sulfur-35  radioactive
protein outer coat
- Two types of bacteriophage were
allowed to infect E. coli bacteria
- Bacteria infected with P32
bacteriophage had radioactivity
inside their cell walls, bacteria
infected with S35 bacteriophage
didn’t  DNA was genetic
material

Bases
- Purines = double ring structure
o Adenine
o Guanine
- Pyrimidines = single ring structure
o Thymine
o Cytosine

DNA Structure
- Numbering of carbon atoms
- Each strand composed of backbone of alternating
deoxyribose and phosphate molecules
o Held together by covalent bonds (phosphodiester
linkage)
o Between OH group of 3’ carbon of deoxyribose
and phosphate group attached to 5’ carbon
o Leaves 5’ carbon and 3’ carbon free at each end
- Backbones antiparallel (5’ – 3’ vs 3’ - 5’)
- Purines pair with pyrimidines
o AT = 2 H bonds
o CG = 3 H bonds

DNA Packaging
- Nucleosomes
o Consists of two molecules each of four histone proteins (so 8 histone proteins),
forming the nucleosome core
o DNA wraps twice around these eight protein molecules
o Fifth type of histone protein helps maintain the nucleosome

DNA Sequences
- Only 2% of DNA is coding DNA (codes for proteins)
- Rest of DNA:
o Acts as regulators of gene expression
o Introns (junk DNA)
o Telomeres
o Codes for tRNA
- Highly repetitive sequences
o Usually comprised of 5-300 base pairs
o Clustered repetitive DNA = satellite DNA
o Transposons = repetitive sequences that can change position within the DNA
(regulators of gene expression)
- Protein coding genes
o Genes that have coding functions
o Base sequence carried from nucleus to ribosome by mRNA
o Composed of exons (coding) and introns (non-coding)
- Structural DNA
o Highly coiled DNA
o Does not have a coding function
o Occurs around centromere and telomeres (ends of each chromosome)
- Short tandem repeats (STRs)
o Short (2-5 bases) repeating sequences of DNA
o Most DNA is identical, repeating sequences that show variation between people
= polymorphisms
o Polymorphisms used for DNA profiling
o Scientists use 13 different loci for DNA profiling called STRs

DNA Replication
- Begins at sites called origins of replication
o Prokaryotic DNA has single origin
o Eukaryotic DNA has thousands of origins
- Summary:
o Begins at origin which appears as a bubble (unzipping catalyzed by helicase
breaking H bonds)
o At the end of each bubble, there is a replication fork (where double-stranded
DNA opens to provide two parental DNA template molecules)
o Bubbles enlarge in both directions; replication process is bidirectional
o Bubbles fuse to produce two identical daughter DNA strands
- Elongation of a new DNA strand
o Primer produced under direction of primase at replication fork
 Primer = short sequence of RNA (5 – 10 bases long)
 Primase allows joining of RNA nucleotides that match exposed DNA bases
at point of replication
o DNA polymerase III allows addition of DNA nucleotides in 5’ to 3’ direction to
produce growing DNA strand
 Nucleotide = dNTP (deoxynucleoside triphosphate)
 Contains deoxyribose, nitrogenous base, and 3 phosphate molecules
 Loss of 2 phosphate molecules  energy provided for bonding to occur
o DNA polymerase I removes primer from 5’ end and replaces it with DNA
molecules
- Antiparallel strands
o Strands run in 5’ to 3’ and 3’ to 5’ direction  antiparallel
o DNA polymerase III assembles in 5’ to 3’ direction on daughter strand
o Leading strand and lagging strand assembled concurrently
o Lagging strand forms more slowly, involved Okazaki fragments, and DNA ligase
 Leading strand formed from 5’ to 3’ continuously towards progressing
replication fork
 Lagging strand assembled by Okazaki forks moving away from
progressing replication fork (5’ to 3’)
 Primer, primase, and DNA polymerase III required to begin formation of
each Okazaki fragment and to begin formation of leading strand
 Primer and primase only needed once for leading strand
 DNA ligase attaches sugar-phosphate backbones of fragments to form a
single DNA strand
- Replication proteins

- DNA replication is extremely fast (~4000 nucleotides/second)


- DNA replication is very accurate

The Human Genome Project


- Involved sequencing of DNA
- Utilized DNA copied through PCR

Topic 7.2 – Transcription and Gene Expression


Central Dogma of Molecular Biology
- DNA  RNA  Protein
o First arrow = transcription, second arrow = translation
- The basic idea that genetic information flows in this direction

Transcription
- RNA polymerase separates two DNA strands,
allows polymerization of RNA nucleotides
o 5’  3’ direction
- DNA strand that carries genetic code is sense
strand (coding strand) vs. antisense strand
(template strand)
o ANTISENSE strand is copied

- Transcription = initiation (promoter)  elongation (transcription unit)  termination


(terminator)
o Promoter region for a particular gene determines which strand is antisense
o RNA polymerase attaches to promoter region  DNA opens and transcription
bubble forms

o Elongation = NTPs used  energy release of two phosphates + RNA polymerase


 polymerization
o Transcription bubble moves from DNA promoter region to terminator
o Terminator = sequence of nucleotides that causes RNA polymerase to detach
when transcribed  mRNA detaches
o In eukaryotes, transcription continues beyond terminator for a significant
number of nucleotides before being released

Post-Transcription Modification of mRNA in Prokaryotes


- Most regulation of gene expression done at transcription
o Introns not present
- Transcription and translation coupled in prokaryotes
- Post transcriptional modification of mRNA does not occur in prokaryotes

Post-Transcription Modification of mRNA in Eukaryotes


- First RNA formed = pre-mRNA or primary RNA transcript
o Contains introns and exons
o Introns = non-coding DNA that need to be spliced (removed)
- 5’ cap and poly-A tail added
o 5’ cap = modified guanine nucleotide with 3 phosphates
o Poly-A tail = 50-250 adenine nucleotides
o Protect mature mRNA from degradation in the cytoplasm
- Spliceosomes composed of small nuclear RNAs (snRNAs) remove introns
o Attach to the intron/exon boundary
o Cause intron to curl up
- When introns removed, exons can be rearranged  more possible proteins

Nucleosomes and Gene Expression


- Nucleosomes  DNA can condense
- To tail of histones in a nucleosome:
o Addition of acetyl group
o Addition of methyl group
o Addition of phosphate group
- E.g.:
o Lysine on histone tails has positive charge, DNA negative  Opposite charges 
condensation of DNA  inhibits transcription
o Adding negative acetyl group to lysine cancels positive charge  less condensed
 transcription can occur

Methylation and Gene Expression


- Methylation  stops/slows down gene expression
- So, high methylation  inactive DNA
- E.g. one of the female X chromosomes is heavily methylated  inactive
- Methylated DNA stays that way through many divisions
Proteins and Gene Expression
- Transcription factors
o Proteins that assist RNA polymerase to bind to promoter region  faster gene
expression
- Transcription activator
o Causes looping of DNA  shorter distance between activator and promoter
region  transcription
- Repressor proteins
o Bind to segments of DNA called silencers to prevent transcription

Environment and Gene Expression


- People with same genotype may express different phenotypes in different
environments
- E.g. production of skin pigmentation during exposure to sunlight

Topic 7.3 – Translation


Ribosomes
- Made up of large and small subunit
- Both made of rRNA and proteins
- Decoding of mRNA is done in the cavity between the subunits
- In cavity, there are 3 binding sites
o E – exit site; site from which tRNA leaves once it loses its amino acid
o P – site that contains tRNA carrying growing polypeptide chain
o A – site that holds tRNA carrying the next amino acid

Translation: RNA  Protein


- Involves several phases
o Initiation
o Elongation/translocation
o Termination
- Genetic code is:
o Degenerate – each amino acid may be associated with more than one codon
o Universal – all organisms share the same genetic code (what base order  what
animo acid)
 Allows for exchange of genes between different organisms