BIO130 Readings

Lecture 2: Chapter 1, Chapter 2 p.p.116-117

Lecture 3: Chapter 2 p.p. 51-64, Chapter 3 p.p. 125-152
Lecture 4: p.p. 202-206, p.p. 208-218
Lecture 5: Chapter 5 p.p. 263-276, p.p. 281-287
Lecture 6: Chapter 5 p.p 276-288, p.p. 292-304
Lecture 7: Chapter 6 p.p. 329-345
Lecture 8: Chapter 6 p.p. 339-358 (3’ processing section)
Lecture 9: Chapter 6 p.p. 366-390

LECTURE 1:

What is going to be taught:

Cellular and genomic diversity
o Prokaryotes vs. Eukaryotes
 Diffs and sims
 General organisation
 Origins
Model organisms: such as E. coli, C. elegans, M. musculus, Drosophila, Arabidopsis

The Molecules of Life:

General concepts about
o DNA, RNA, proteins;
 their nomenclature, important structural components, bonds that
hold them together

DNA
- nuclear and organellar genomes (mitochondrial and chloroplast DNA vs. nuclear)
- they perform different tasks and code for different proteins
o mito and chloro have their own DNA because they were originally two
organisms, one engulfed the other, and now they are endosymbiotic; one
organism living inside another
- chromosomes and chromatin
o chromatin contains genes that modify gene expression in cell
o how DNA is packaged and then unwrapped to be available for expression
- DNA replication and repair
- Genes and genomics

RNA
- general characterisation of transcription
o exons, introns, synthesising different kinds
- RNA processing
- Transcriptomics – all transcripts and proteins being expressed at one type
(everything being transcribed – not [introns]****)
Proteins
- translation
- post-translation processing
- proteomics

LECTURE 2: Introduction to Cells and Diversity

Covered: Prokaryotic vs. Eukaryotic cells, Diversity and Model Systems, Origins,
Introduction to Nucleic Acids

Tree of Life: shows how three domains of life (bacteria [eubacteria], archaea
[archaebacteria], and eukaryotes)
Some examples:
- methanothermobacteria – anaerobic, low pH (acidic) conditions (cow stomach)
- cyanobacteria – capable of photosynthesis, found in soil
- methanococcus – found in hydrothermal vents at bottom of the ocean
Chart has a scale showing 1 change/10 nucleotides (n) showing HOW different they are

Two Main Types of Cells:

Prokaryotes: no nuclei, single-celled organisms (e.g. eubacteria and archaea)

- has selectively permeable cell wall and plasma membrane
- flagellum for motion (not all have this)
- nucleoid: an irregularly shaped region in the cell w/ unprotected genetic material
- ribosomes for protein synthesis in cytosol
Eukaryotes: nuclei, single or multicellular, a lot larger w/ cytoskeletons for structural
integrity (plants, fungi, animals, humans)
- contains endomembranes, separating cell into functional compartments
(organelles)
- microtubules, centrosome w/ pair of centrioles
- Nucleus w/ nuclear envelope containing DNA
- Extracellular matrix and lysosome NOT found in plant cells
- Mitochondria produce ATP
- **Vacuoles do not exist in animal cells, are exclusive to plants

ENDOSYMBIOSIS: Origins of Mitochondria

1. Anaerobic ancestor engulfed an aerobic bacterium producing ATP from oxygen

2. Mitochondria escaped digestion, and was protected by the cell, providing energy,
and containing its own DNA
3. Same story with chloroplasts and some algae
Didinium: carnivorous, single-celled eukaryote that has cilia and shoots poison darts into
its target and engulfs it
Model Organisms: species that have been studied intensively for a long period of time
and are models for deriving fundamental biological principles (e.g. drosophila)
Attributes:
- rapid development, short life cycles
- small adult reproductive size (not an elephant)
- readily available
- tractability – ease of manipulation or modification
- understandable genetics – simple organisms

A Model Prokaryote: E. coli – heterotrophic eubacterium in human gut

Synechocytis – free living phototrophic cyanobacterium

A Model Eukaryote: Yeast – minimal eukaryote model

Arabidopsis – a model flowering species
Caenorhabditis elegans – nematode worm with 959 body cells
Drosophila, mice, humans

Central Dogma: information flow in the cell

DNA ---------------- RNA -------------- Protein

Transcription Translation
RNA synth protein synth

*Note DNA doesn’t actually turn into protein or RNA

Transcription Overview:
DNA, RNA, and protein are all linear chains of information
Information in nucleic acid sequence is translated into an amino acid sequence via a
genetic code which is essentially universal (all cells have the same one) among all
species. It is also degenerate and semi-conservative.

Genetic code: refers to the codons (a triplet of nucleotides) that correspond to a specific
amino acid. It is degenerate because many different codons code for the same AA.
CC“X” – Proline, UAA, UAG, UGA – stop codon, AUG – start codon

Consequences of degeneracy:
1. All 20 AAs are coded by the DNA
2. There are opportunities for incorporation of new codons or AAs
3. Base-pairing between codon and anti-codon structures occurs more easily
4. Random mutation resulting in AA changes are reduced, with less damage done

Nucleic Acids: the genetic material of a cell; an organism’s blueprints

Contains: - pentose sugar, a scaffold (railing) for bases
- nitrogenous base, which varies (ACTG)
- phosphate group, the backbone (1, 2 or 3 Ps)
One monomer is all of these (a nucleotide)
Purines: Adenine, Guanine
Pyrimidines: Cytosine, Thymine, and Uracil

DNA: deoxyribose sugar used, ACTG bases (thymine has an extra methyl group)
RNA: ribose sugar used, ACUG bases (ribose has an extra O)

Nucleic Acid Nomenclature

1. Nucleioside monophosphate (e.g. AMP – adenosine monophosphate)
a. Sugar + base + 1P
2. Nucleioside diphosphate (e.g. ADP – adenosine diphosphate)
a. Sugar + base + 2P
3. Nucleoside triphosphate (e.g. ATP – adenosine triphosphate)
a. Sugar + base + 3P
A nucleoside (no phosphates) is a base plus a sugar
A nucleoTIDE is a base, sugar, and at least 1P
e.g. a nucleotide is ADENINE, a nucleoside is ADENOSINE
*others are: Guanidine, Cytodine, Thymidine, Uracine
*nucleoside + sugar = -ina (cytodina)

DNA is a template for the three main types of RNA involved in transcription:
messengerRNA (translation): protein
transferRNA: transportation of amino acids for protein synthesis
ribosomalRNA: part of the ribosome

Genome: all cells or organism’s genetic sequence

Transcriptome: all RNA species in cell
Proteome: all protein in cell
Interactome: all interactions in proteins or molecules
Metabolome: all small molecule metabolites, or organic molecules (HORMONES)
Phenome: complete set of phenotypes (all of the above)

LECTURE 3: Introduction to Nucleic Acids and Proteins

Molecular Interactions: interactions between individual molecules usually mediated by

noncovalent attractions. Phosphodiester bonds are those between phosphate and sugar in
DNA. They are very stable and permanent
Electrostatic attractions: ionic bonds within proteins due to high level of water in
cell preventing charges from happening
Van der Waals attractions: bringing together macromolecules
Hydrophobic forces: hydrophobia causes molecules to fold into each other

Individually they are very weak, but can sum to generate strong binding between
molecules

The amine end (N terminus) of a protein is like the 5’ end, and the carboxyl end is the 3’
(C-terminus)

Nucleic acid chains:

DNA is synthesised from dNTPs (deoxyribonucleoside triphosphates)
RNA “ “ “ NTPs (ribonucleoside triphosphates)

Base Pairing: A-T make 2 H-bonds, C-G make 3 H-bonds

Structure: Major groove (larger segment) allows for access by proteins like modifying
enzymes, with the bases.
The strands are antiparallel, one is 5’-3’, the other is 3’-5’. At the 3’ end is a hydroxyl
group (OH) off the sugar, and the 5’ has a phosphate (-PO4).
High temperatures or pH will denature the DNA, and be unzipped. Bringing them back
to normal conditions will cause them to reanneal and nucleotides bonds will reform due
to complementary base pairing. This is a reversible process, the denaturation temperature
is call Tm, and varies between species.

Polymerase Chain Reaction:

1. Heat to separate strands
2. Hybrization of primers pieces of DNA synthesised 15-20n complementary to gene
wanted for study
3. Add DNA polymerase to add to existing fragments, making the sequence for gene
of interest
4. DNA synthesised from primers

Introduction to Protein Structure:

Primary (sequence) Amino acid sequence
Secondary (local folding) alpha helix, beta sheet
Tertiary (long-range folding) 3-D structure
Quaternary (multimeric organization) Multiple polypeptide chains
Supramolecular (large scale assembly) Large scale protein assemblies
`
Proteins are composed of amino acids
The amino side-chains or “R” group is variable, and determines the type of AA
4 basic categories: basic (+ve), acidic (-ve), uncharged polar (hydrophilic), non-polar
(hydrophobic) – these are consequences of the R groups

Cysteine can form Cys bridges across a chain of AAs

Mutational steps: # mutations between codons to turn them into a new AA
# to get from proline to cysteine: 2, max 3

Groups with similar properties are clustered in the codon table, and have fewer
mutational steps between them.

Order of AAs is important, i.e. Leu-Enkephalin, which modulates perception of pain –

causes sense of wellness when running for example. We always right N terminus to C
terminus.

Alpha Helix: 3.6 AA per turn, girder like, steel frames, can create pores through
membrane walls allowing certain molecules in based on the R groups
Beta Sheet: runs antiparallel – H-bonds are formed between strands, like plywood,
forming a wall

H-bonding in secondary structures

Which atoms are H-bonded? Carbonyl oxygen, amide hydrogen
Alpha helices bond between 4 AAs apart within the same strand
Beta sheets between AA in different strands

The alpha helices can have amphipathic qualities, wherein certain R groups will be on
one side of the helix that are hydrophobic, and the other hydrophilic. Useful for when it
crosses a pore leading into a cell
- creates “supercoil”, or coiled coil, where polar goes with polar and v.v., coiled in
again on its self

Tertiary bonds: hydrophobic interactions, non-covalent bonds, covalent disulphide bonds

Quaternary structure: Haemoglobin formed from different subunits, 2 alphas and 2 betas,
and each is a polypeptide

LECTURE 4: Genomes and Chromosomes

Neanderthal Genome Sequence:

 - 65% known
- DNA recovered from 38 000 year old bones in Croatia
- Modern humans w/ non-African ancestral genomes share 1-4% DNA
o Bone and skin are tougher (no clothes)
o Ribs wider and smaller
- Partial sequence analysis shows only 78 AA variants in protein coding genes that
MIGHT impact function

Human Genome: 3 billion bps/genome, one maternal + one paternal = you

- ~25 000 genes spread across 23 chromosomes
- Each group of 23 chromosomes from either parent is a genome .: Two genomes
- Chromosome: an organised structure of DNA and protein
- Genomes can be any size – mitochondria just 16.6 kb, chloroplast is 160 kb
o So small because most coding is done by DNA, they no longer need genes
to live solo, proteins made by DNA move to mitochondria
- Prokaryote DNA is generally smaller, can sequence in hours
- However, an amoeba has more kb than us – size doesn’t correlate to
complexity!

1.5% of genome encodes protein – most is non-coding or redundant, 50% is repetitive

Transposons:
- LINE: long interspersed nuclear elements
- SINE: short “ “ “
- Retroviruses: stick their genetic material into your genome
- Transposon fossils
Repeated sequences: all of above plus simple sequence repeats, segmental duplications
Unique sequences including: non-repetitive DNA neither in introns or codons, nothing to
do with making RNA or proteins
Genes: introns and protein coding regions

Packaging of DNA in Prokaryotic Cells:

DNA is condensed 1 000 fold, DNA + protein form the nucleoid
Components that help packaging include:
- +vely charged polyamines (proteins), react to the –ve DNA
- NAP (nucleoid-associated proteins) such as H-NS, histone-like proteins HU, IHF,
FIS
- Supercoiling of DNA by topoisomerase, wind and unwinding DNA

Eukaryotic Cells: 6 billion bp/cell, around 2 metres, nucleus is 6 micrometres

Solution: packaged as chromosomes – karyotype: artificial array of paired chromosomes
in numerical order, a diagnostic aid
Chromatin: single, long, linear DNA and associated proteins
- is packaged tightly, but DNA must be accessible for transcription, replication,
repair
- Dynamic
Nucleosome (eukaryotic!!): basic structural unit of chromatin – think beads (proteins)
on a string (DNA), linker + wrapper + core protein (histone), histone + wrap =
nucleosome core, the wrapping requires ATP
Components:
- linker DNA 80 bps long
- wrapper DNA around histones ~147 bps long
- histones: small proteins +vely charged; stick well to DNA
o four core histones: H2A, H2B, H3, H4, with a pair of each in octamer core
o one linker histone in on linking part (H1), clips DNA in place on bead
Heterochromatin: during M phase of division, includes mitotic and meiotic
chromosomes, centromeres (centre of chromo.) and telomeric regions (end), one of Xs in
females. In these locations gene expression is suppressed – no transcription!
Euchromatin: (looser, more access) where genes tend to be expressed, covalent
modification of histones, RNA polymerase and other allow for the switch between these
two forms.

LECTURE 5: Introduction to DNA Replication

Replication is semiconservative – each daughter cell has a template strand and novel
strand
Always goes 5’ to 3’ ends, with three models:
- Unidirectional growth from two starting points, e.g. linear viruses
- Unidirectional growth of two strands from one starting point (some plasmids)
- Bidirectional growth from one starting point, e.g. eukaryotes & bacteria
Always starts at easy to open A-T rich segments (only 2 H-bonds) which are recognised
by initiator proteins – TFII and the TATA box for example
- single origin point: bacteria
- multiple: eukaryotes with many origins of replication

Ex. Budding yeast with and without Autonomously replicating sequences (ARS)
A plasmid vector is inserting with histidine producing gene, w/ and w/out ARS. Ones w/
ARS survive, ones w/out die because His gene is not replicated into future generations.

In bacteria: circular genomes are pulled apart and nucleotides added 5’-3’, looks like an
eye opening up – points where parental strands are being pulled apart called replication
forks.
Leading strand: 5’-3’ going towards direction of separation
Lagging strand: filled in based off the leading strand with Okazaki fragments. Between
fragments are “nicks” that need to be covalently bonded; replication is towards 5’.

Ingredients for synthesis: Origin, ATP, primers, DNAases, DNA polymerase, accessory
proteins
1. Origin of replication
2. Binding of initiator proteins: bind to origin, help helicase bind to initiator
proteins, requires ATP
 3. Unwinding by helicase: Has 6 subunits, unwinds in 5’-3’ along lagging strand
template, requires ATP
4. Binding of single-strand binding proteins: keeps DNA separated to prevent H-
bonding by binding ssDNA, preventing hairpins and kinks.
5. RNA primers made by primase: to begin DNA polymerase requires bound
primase to synthesise an RNA primer with 3’ OH end to start binding to incoming
nucleotides; goes 5’-3’
6. DNA polymerase: attaches incoming dNTPs (deoxyribonucleoside triphosphate);
has polymerizing and editing sections.
7. Sliding clamp holds polymerase onto DNA
8. Nick sealing between Okazaki fragments by DNA ligase: DNA repair system
removes RNA primer and replaces it with the correct sequence

LECTURE 6: DNA Replication II

1. Leading strand synthesised continuously from single RNA primers

2. Lagging strand discontinuously from multiple primers
3. Okazaki fragment = RNA primer + DNA
4. Goes 5’-3’, primosome = helicase + primase
5. Helicase is located on the lagging strand

At ends of chromosome, the 5’ end, information (telomeres) is lost due to removal of the
primers without a way to add more nucleotides behind the last primer. This is a problem
for the lagging end – telomeres exist here to fold back into the same strand and protect
coding information.
Telomerase: generates G-rich ends, adding nucleotides to the 3’ end of lagging template
- cancer cells have high levels of telomerase

Supercoiled in the same direction as DNA = + supercoils, opposite = - supercoils

Replication uses + supercoils

Stress released by Topoisomerase type I: breaks single-stranded DNA, allowing it to

rotate around the sugar phosphate group of one strand
Topoisomerase type II: untangle and separates double stranded DNA (i.e. two plasmids
that are separated), breaks double-stranded to allow one double-stranded helix to pass
through the other

DNA Proofreading:

RNA polymerases have 1 in 10^4 error rate, DNA polymerases 1 in 10^9

Two proofreading methods:
1. 3’ to 5’ exonuclease: chews back misincorporated nucleotides – a literal backspace
button – DNA polymerase engages in editing in a different site than its does
polymerization
5’-3’ direction allows for efficient error correction and chain growth
2. Strand-directed mismatch repair: post-polymerase repair process, initiated by
detection of distortion in the geometry of the helix by mismatched base pairs

- DNA can be damaged after synthesis by oxidation, radiation, heat or chemicals

- defects in repair mechanisms can cause breast and skin cancers
3. Base-excision repair: targets individual incorrect nucleotides

LECTURE 7: Transcription I

LECTURE 8: Transcription II

Introns are transcribed but not translated, exons are transcribed and may or may not be
translated

M, t, and rRNAs code for protein

Three types of eukaryotic RNA polymerases (RNAPs) required for transcription:

1. RNA polymerase I – 5.8S, 18S, 28S RNA genes (larger S = larger RNA)
2. “ “ II – all protein coding genes (mRNA)
3. “ “ III – tRNA genes

RNAPs are complex structures with many subunits, some are common to all three, some
resemble those of bacterial RNAPs – eukaryotic requires more proteins called “general
transcription factors” because they have to deal with unravelling and coiling

Eu vs. bacterial RNAPs:

Eu requires proteins to help position them at promoter called transcription factors,
which fulfill similar role to sigma subunit of bac RNAPs. Eu RNAPs also deal with
chromosomal structure, nucleosomes, etc.
- No introns in bacterial gene structure
- Eukaryotic mRNA has guanine cap and poly-A tail.

Promoters:
+1 = transcription starting point, “Upstream” are –ve #s, not transcribed, “downstream” is
where RNAP starts making mRNA, which ARE transcribed.

1. TATA box: is highly conserved, 25-36 bp upstream of start point, positions

RNAP II
2. TFII is the transcription factor for RNAP II, TBP is TATA binding protein

Initiation of transcription: RNAP II ACTIVATED THROUGH

PHOSPHORYLATION****
1. TBP is a subunit of TFIID in highly transcribed genes, binds to TATA box,
bending and distorting the DNA to shape it better for RNAP II
 2. TFIIH: large, 9 subunits; ‘H’ is for helicase, opens up DNA strands and requires
ATP to pry apart DNA strands at start point
3. TFIIB: binds to BRE element helps position RNAP II
4. CTD: carboxyterminal domain
5. All of above are “Transcription initiation complex”
6. Complex begins doing “abortive” (stop and start) transcription
7. TFIIH comes in, “kissing”, then makes it transcribe at high speeds; also
phosphorylates the 5th Serine of CT tail, so that transcription can begin
a. Histone modifying enzyme: covalently modifies the parts

RNA Processing:
1. Add 5’ pre-mRNA cap – removes 1 P, adds GMP (guanidine monophosphate),
and adds methyl group to G base  guanine becomes first ribose
2. Guanine cap is added to the 5’ end of transcript, making a 5’-5’ linkage
(BACKWARDS), enzymes involved: phosphatase, guanyl transferase, and
methyl transferase.
3. Prevents exonucleases from chewing the transcript

Pre-mRNA splicing reaction: (splicing proteins remove introns & some exons)
1. Adenosine attacks the 5’ splice site
2. 3’ of one exon reacts w/ 5’ of next exon to release introns – they merge
3. The introns forms a lasso shape around adenosine called LARIAT
4. Process requires the 2’ OH group, .: cannot happen in DNA (deoxyribose)

RNA and protein interactions for splicing: snRNA = small nuclear RNA
- snRNPs = snRNAs + proteins
- Pre-mRNA splicing is performed by the spliceosome, ATP-dependent, binds to
many nucleotide sequences

LECTURE 9: Translation

- codons are read as triplets

- DNA encodes for all 20 AAs
- There is redundancy – multiple codons for most AAs
- Reading frames define the AA sequence
o If you move even one nucleotide over, it changes the entire AA chain

tRNA: ~80 nucleotides, each triplet in the anticodon region is antiparallel and
complements the codon of the mRNA
- some tRNA can recognise more than one codon

Ensuring fidelity: the right tRNA has to have the right AA on it

- tRNA synthetase puts the right AA on the right tRNA
o similar to DNA polymerase, uses a backspace method for chopping
incorrect units by hydrolytic editing
 o identifies tRNA anticodon nucleotides, recognises nucleotide sequence of
acceptor stem, reads nucleotide information at additional position on the
tRNA
- complementary base pairing

Ribosomes: Eukaryotic is larger than prokaryotic

- located on endoplasmic reticulum (eukaryotes only – pros don’t have ER) and
cytosol

Protein Synthesis

A-site: Aminoacyl-tRNA
P-site: Peptidyl-tRNA
E-site: Exit

Since protein synthesis is slow, polysomes, or polyribosomes occur – many ribosomes

spaced every 80 nucleotides are reading the mRNA and making the requisite proteins

Quality Control: GTP w/ EF-Tu (elongation factors) I escort aminoacyl-tRNA to

ribosome and checks for correct AA and anticodon. Also holds tRNA away from the P-
site to prevent premature peptide bond from forming. Later, EF-Gmoves the small
ribosomal unit (bottom one) quicker so the codon nucleotides don’t lag behind.

Elongation factors:
- speed up and make more efficient the process of protein synthesis
 - mediated by EF-Tu, and GTP hydrolysis
- EF-Tu binds aminoacyl-tRNA
- EF-G helps ratchet forward one codon

Prokaryote ribosome binding site are also called “Shine-Dalgarno sequences”, and have
multiple initiation sites

Termination of translation: Human translation release factor

- molecular mimicry – looks like a tRNA, but is a protein

Proteins fold as they exit the exit of the ribosome, happens at the same time as translation
When things go awry, protein chaperones (Hsp60, Hsp70) catalyse the proteins to make
them fold properly – if that doesn’t work, a protease destroys the useless protein