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xThe Changing Genome
Transgenesis is the stable introduction of a gene into an organism
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However, the
expression is mostly used in reference to multicellular organisms
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Due to the universality of the genetic
code, the gene could be from a different species - e
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the introduction of a fish gene to
convey cold hardiness in tomatoes
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Gene knock-outs have been very useful in model organisms for gaining an understanding of
gene function
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This can sometimes result is death before birth or compensatory
up-regulation of other genes
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It could also mean changing
a genetic variant that causes disease into the wildtype allele
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It allows the understanding of the function of genes that have been knocked down without
having to worry about whether it is lethal
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Transgenesis in C elegans via microinjection;
1) Transgenes are added through the microinjection of foreign DNA into the gonad
2) DNA for a dominant mutant allele of a gene that causes worms to move in a
corkscrew pattern as well as a dye are also injected
3) Inside the cell, injected DNA molecules are then joined together in a long strand,
which is maintained during mitosis and meiosis
The Drosophila P element;
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Absent from original laboratory stains but present in wild fruit flies
A cross between a wild-caught male and a lab strain female gives a normal F 1
generation, but shows defects in subsequent ones (hybrid dysgenesis)

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It became a way of producing new mutant strains
A gene in the P element, transportase, is not necessary for its function, and can be
replaced with a transgene that can be introduced

Plant Transgenesis;
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Parasites can cause crown gall in plants
Bacterium carries plasmid which integrates into the host genome and causes a
tumour by expressing plant growth hormones and the amino acid opine
Opine provides nourishment for the bacterium and is relatively harmless to the plant

Plant transgenesis utilises naturally occurring gene transfer process
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The selectable marker such as an antibiotic is
added
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This same method is used for transfecting food crops with transgenes to make them
resistant to parasites, herbicides or for additional nutrients
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It
allows the DNA modification to be targeted a specific cell type or be triggered by a specific
external stimulus
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jax
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Cre will excise “floxed” genes
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A transgenic mouse which expresses Cre, specifically in the heart but is in the absence of a
floxed gene means that it has no overall effect on the cells
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Furthermore, in the absence of Cre, the
floxing of the gene will make no difference in any other cell
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It does this by creating loops of fiber, to pack
the DNA more densely
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Gene Content;
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Around 20,000 protein-coding genes
Around 13,000 pseudogenes
1,756 microRNA genes
Around 5,200 other non-coding RNAs

This means that there is on average 1 gene for every 150,000 base pairs of DNA
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One chromosome has half the normal gene content, others can have three times as many
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Deep sequencing of RNA suggests that more than
half of the human genome is actually transcribed at low levels meaning that something
happens which is not completely known
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Introns are integral to gene expression regulation
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Alternative splicing is widely used to generate multiple proteins from a single gene
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Exons contains the information required to encode a protein
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Introns can then be removed to
make a functioning messenger RNA that can be translated into a protein
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A constitutive or housekeeping gene is expressed in all cells all the time
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Depending on the cell type and
conditions
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Normal Variation and Personalised Genomics

There are four main sources of variation;
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Purifying selection
Purifying selection prevents harmful mutations from establishing themselves in the
population and becoming common polymorphism
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However, harmful
dominant alleles that cause severe symptoms after the carrier has had children, can
remain
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It is a genome sequence evolution as a
result of an advantageous mutation that increases the fitness of the population in its
environment
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Founder effect
Occurs when a small group of individuals move to a new location and is caused by
there only being a small sample of the genetic variation in the founder population
being brought into the gene pool, therefore causing a new bias in allele frequencies
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Determining Pigmentation;
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Eye, hair and skin pigmentations have all been found to be in the polygenic traits

Height Genetics;
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Height has one of the highest heritability estimates of all human traits
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High
heritability does not equate to large effects of single genetic polymorphisms in
specific genes
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A classical sequencing is time
consuming and requires relatively high amounts, so, modern methods are preferred
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However, it cannot copy a DNA strand so long as it is joined to its complementary
strand
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Sanger sequencing is based on the extension of a primer
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The temperature is then increased
again allowing DNA polymerase to synthesize new DNA starting from the primer
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At that point, no further nucleotides can be added so the
strand will end
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This results in a series of newly
synthesized strands, all of which end in the same ddNTP
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Majority of human tumours arise from epithelial
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Carcinogens e
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radiation, chemicals, virus infection etc are causes of cancers
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P-450 pathway is normally used by cells to remove noxious
substances but can also activate indirect carcinogens
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There are three broad categories of genes implicated in cancer;
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Proto-oncogenes
Tumor-suppressor genes
Genome maintenance genes - mutation allows propagation of gene mutation as the
DNA repair is insufficient
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Mutation of these proto-oncogenes are called gain-of-function mutations, they gain on top of
their baseline functions as a result of mutation and are known called oncogenes
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The mechanisms of gain-of-function mutation include;
point mutation (change in a single base pair), chromosomal translocation (fuses two genes
together to produce a hybrid) and amplification (generation of numerous proto-oncogene
copies exist, leading to overproduction of the encoded protein
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An example is; a
mutation in HER2 receptor transmembrane region causes dimerization and constitutive
activation of the receptor, even in the absence of normal EGF-related ligand
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Trastuzumab, a monoclonal can target Her-2 leading to the
repression of; Her-2 mediated growth signalling and also the destruction by the immune
system
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Transcription factors can drive cancers; prostate cancer is stimulated by testosterone,
testosterone activated the androgen receptor
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AR and ER are examples of nuclear hormone receptors
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Elements within the DNA are found in target
genes
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Enzalutamide, inhibits androgen binding to AR, it then inhibits nuclear translocation of the
AR
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This is important because, the
assay can be done to simply monitor the amount of PSA in the blood
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Genes can fuse together to drive cancer
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BCR-ABL genes code for a driver kinase, fusion protein can
phosphorylate and activate survival pathways
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Viruses can also cause cancer
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The virus can cause cancer by inhibiting tumor suppressor
gene function
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As long as they are intact, cancer cells should not survive
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Rationale for existence - controlling cell-cycle checkpoints and development
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The loss-of-function mutation deletes an important brake on the cell cycle P53is a tumour
suppressor and can be activated to stop the cell cycle and give the body more of a chance to
suppress the cancer cells
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Cells with p53 become arrested in G1 phase if treated with DNA
damaging agents
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Retinoblastoma 1 is a tumour suppressor and functions by acting as a negative regulator of
transcription factor E2F
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RB traps E2F transcription factor when unphosphorylated, preventing activation of
many genes required for DNA synthesis
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In S phase, Rb becomes completely phosphorylated and dissociates E2F to
turn on genes required for DNA synthesis
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In normal cell oxygen determines the destiny of
pyruvate
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Cancer can be hit with a metabolite mimic;

1
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3
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2-hydroxyl group of glucose can be replaced by hydrogen, so no further glycolysis
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Cells with higher glucose uptake, have higher uptake of 2-DG
6
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Mutations in isocitrate dehy drogenase are very common in
cancers
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Tumour growth requires angiogenesis
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A tumour without blood supply can only grow into a
mass of 1 million cells
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VEGF targeting agents can block tumour angiogenesis and are being exploited for cancer
treatment
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Under normoxia, the hormone is synthesized by the inner cortex
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HIF-1
alpha is a key transcription factor induced by hypoxia and regulated by a proline hydroxylase
pathways that are regulated by hypoxia and angiogenesis tissue invasion and metastasis
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Genetic instability can lead
to genetic divergence or cancer, hereditary disease
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DNA polymerases introduce copying errors
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coli it introduces 1 wrong base for every 10k bases incorporated, but
the mutation rate is 1 wrong base per 1 billion
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Point mutations are common
events, they change in one base of the DNA and most commonly occur in the spontaneous
change of DNA
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Chemical and radiation changes can damage DNA and cause mutations of their own
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Loss of information results in the inhibition of
DNA replication leading to mutations
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Cytosine and 5-Methylcytosine
are most common deamination reactions e
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Deamination can lead to a G:C base pairing
changing to an A:T
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DNA damage can be repaired
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This can lead to the activation of one of the excision repair pathways
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DNA DSBs are highly
toxic, a few sustained DSBs are enough to kill a cell
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However, the incorrect joining of
DSBs can created hybrid genes, and can place a low expression gene under the control of a
strong promoter
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2) Apurinic endonuclease recognises and cuts the DNA backbone at the abasic site,
creating a 5’ hydroxyl
3) Apuric lyase associated with DNA polymerase B removes the abasic deoxyribose
phosphate
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5) DNA ligase forms a phosphodiester bond to seat the nick
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The endonuclease that cuts the daughter
strand
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A DNA exonuclease removes several nucleotides
from the cut end of the daughter strand, including the mismatched base
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The loss of mismatch excision repair can cause cancer
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Random mutations in the second
copy predispose to hereditary nonpolyposis colon cancer
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NER proteins slide along the DNA looking for bulges and other distortion in the
DNA
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NER defects can lead to Xeroderma Pigmentosum
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30 proteins execute NER and defect were identified in individuals with XP
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Mutations in XP-A to XP-G cause NER defects
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BRCA is a tumour suppressor, it helps repair DNA double strand breaks and in doing
so protects against breast cancer
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Normally breast cells have intact BRCA and PARP inhibition will not be toxic
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Because breast cells have BRCA deficiency, blocking PARP activity will cause DSB
accumulation due to the lack of BRCA
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RNA polymerase II is a large protein
composed of many different subunits
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The carboxy-terminal domain is important and it acts as a phosphorylation site
which is vital for the regulation of the enzyme
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After binding, it then has to separate the DNA strands and initiate transcription at
the start site
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CpG islands are housekeeping genes, they are found in 70% of genes and are responsible
for lowering transcription rates
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TFIID binds to the promoter
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TAF (TBP
associated factor) are also part of TFIID, and they recruit to promoters which do not have a
TATA-box e
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Initiator and DPE
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TFIIB is a monomer which binds to the DNA on
either side of the TATA-box and also binds to TBP
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When RNA pol II combines with the TFIIF, it is known as the closed preinitiation complex
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TFIIE binds first and creates a docking
site for TFIIH (includes helicase and kinase)
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Transition to transcription involves TFIIH helicase which unwinds the DNA and pushes back
on the promoter bound GTF using ATP to form a transcription type bubble
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TBP/TFIID is
left at the promoter and the other general TFs dissociate
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There are two factors
involved; negative elongation factor and DRB sensitivity inducing factor
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This
switches Pol II to elongation mode, so elongation factors are recruited, making the enzyme
proccessive
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e
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- Typically around 50-200bp, but composed of several functional elements
- They are directionally independent and usually tissue specific
Proximal Promoter Elements;
- Sequences close to TSS, position may affect activity
- Short, around 6-10 bp
- Often directionally independent and tissue specific
A transcription factor is a protein that initiates or regulates transcription in eukaryotic cells
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g
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RNA binding domains are often dimers i
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homodimers or heterodimers
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g
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The alpha helix
inserts into the major groove of the DNA and it is classified into structural motifs (types)
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Zinc-finger proteins are short sequence fold around co-ordinated Zn2+, they consist of side
chains of cysteine and histidine
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They
form a coiled-coil dimerization domain which is stabilised by hydrophobic interactions
between the monomers
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Activation domains are structurally diverse, often rich in one type of amino acid and may
have intrinsically disordered conformation but become more ordered with a co-activator
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There are three basic mechanisms that underpin transcriptional activation;
1
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Recruitment of RNA polymerase via mediator complex or recruitment of general
transcription factors (TFIID)

3
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The first key protein is eIF4E, the
cap binding protein
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Rate limiting steps are the key targets for regulation and deregulation
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The three polymerases are structurally similar to
one another and share some common subunits, but they transcribe different categories of
genes;
Type of Polymerase

Genes Transcribed

RNA polymerase I

5
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RNA polymerase III

tRNA genes, 5S rRNA genes, some snRNA
genes and genes for other small RNAs

Eukaryotic RNA polymerase II has many structural similarities to bacterial RNA polymerase
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While bacterial RNA polymerase requires only a single transcription-initiation factor to
begin transcription, eukaryotic RNA polymerases require many such factors,
collectively called the general transcription factors
2
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RNA Polymerase II Requires a Set of General Transcription Factors;
The general transcription factors help to position eukaryotic RNA polymerase correctly at the
promoter, aid in pulling apart the two strands of DNA to allow transcription to begin, and
release RNA polymerase from the promoter to start its elongation mode
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They
consist of a set of interacting proteins denoted as TFFIA, TFIIB, TFIIC, TFIID, and so on
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Name

Number of Subunits

Role in Transition Initiation

TFIID

1

Recognizes TATA box
Recognizes other DNA
sequences near the
transcription start point;
regulates DNA-binding by
TBP

TFIIB

1

Recognizes BRE element in
promoters; accurately
positions RNA polymerase
at the start site of
transcription

TFIIF

3

Stabilizes RNA polymerase
interaction with TBP and
TFIIB; helps attract TFIIE
and TFIIH

TFIIE

2

Attracts and regulates TFIIH

TFIIH

9

Unwinds DNA at the
transcription start point,
phosphorylates Ser5 of the
RNA polymerase CTD;
releases RNA polymerase
from the promoter

Polymerase II Also Requires Activator, Mediator and Chromatin Modifying Proteins;
Studies of RNA polymerase II and its general transcription acting on DNA templates purified
in vitro systems established the model for transcription initiation just described
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As a result, transcription initiation in a eukaryotic cell is more complex
and requires proteins than it does purified DNA
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Eukaryotic transcription initiation in vivo
requires the presence of a large protein complex known as the mediator, which allows the
activator proteins to communicate properly with polymerase II, and with the general
transcription factors
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Both types of enzymes can increase access to the DNA in
chromatin Both types of enzymes can increase access to the DNA in chromatin, and by
doing so they facilitate the assembly of the transcription initiation machinery onto DNA
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The order of assembly of these proteins does not seem to follow a
prescribed pathway; rather, the order differs from gene to gene
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To begin transcribing RNA polymerase II must be released from this large complex of
proteins
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Transcription Elongation in Eukaryotes Requires Accessory Proteins;
Once RNA polymerase has initiated transcription, it moves jerkily, pausing at some DNA
sequences and rapidly transcribing through others
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These factors typically associate with RNA polymerase shortly after initiation and help
the polymerase moves through the wide variety of DNA sequences that are found in genes
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In addition, histone chaperones help by partially
disassembling nucleosomes in front of a moving RNA polymerase and assembling them
behind
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