MBS 344 Final Exam Slides FA24

MBS 344: Molecular Biology
Final Exam Lecture Slides

General Information on Final Exam
• Final exam will consist of 28 multiple choice (56pts) and 7 short-answer questions (44pts); this exam will cover
All Lectures. Content from papers 1-3 will not be covered.
• Questions will be based on previous exams, practice exams, and study guides. About 25% of the questions on the
final will be from previous exams and practice exams. Short answer questions will not be multi-part questions.
• This will be a paper exam, and you need to be on campus to take the exam. Taking the final as an online exam is
not an option for this course.
• You can use pen or #2 pencil on exam. If you are using pencil, make sure you write dark enough for scanning.
• Exams will be scanned, uploaded to Gradescope and graded by instructor and TAs. Graded exams will not be
published. To view graded final exam, you need to schedule a one-on-one meeting with the instructor.
• To prepare for the final exam, you should look over “Final Exam Lecture Slides”, the final exam study guide, and
previous midterm exams (passive learning).
• You should also try to identify concepts that you don’t fully understand. Print out blank practice exams,
homework assignments, or discussion activities and try to fill them out without your notes (active learning).
DNA Analysis Techniques
Polymerase Chain reaction (PCR)
• Don’t have to clone DNA into a plasmid to amplify a

gene anymore
• PCR can amplify any region of the genome using isolated

genomic DNA and a set of primers: short segment of
single-stranded DNA
• Primers specify the region of amplification by providing

a 3’OH for the DNA polymerase
• PCR can amplify specific regions from very small

amounts of DNA which is useful for researchers,
clinicians and forensic scientists
DNA Analysis Techniques
PCR requires three steps to amplify specific region of DNA:
1. Denature (95°C):high temperature separates DNA strands by

breaking hydrogen bonds between bases
2. Anneal (~55°C):lower temperature allows primers to anneal

to template DNA
3. Extend (~70°C): optimal temperature for DNA polymerase to

add nucleotides to synthesize new strand in 5’ to 3’ direction
Lab Techniques
• These three steps are repeated many times (~35 cycles) to make millions
of copies of DNA
• Agarose gel electrophoresis sorts DNA segments by size; often used to

check if PCR amplification was successful and determine size of PCR
product
• M = molecular weight marker (used to measure size of bands in gel);

1-10 are PCR samples
• Should see one band in gel if PCR was successful
• Gel electrophoresis is used to visual bands and estimate size of bands

Lab Techniques
Quantitative PCR (qPCR)
• PCR amplification can also be detected using fluorescence
SYBR green: fluorescence molecule that binds to double-stranded

DNA (dsDNA). PCR = more dsDNA = more fluorescence
Taqman probe: primer has fluorescence molecule (R) and quencher (Q)
that suppresses fluorescence. Primer binds to single-stranded DNA.
When amplification occurs DNA polymerase cleaves probe and releases
fluorescence molecule from quencher, so fluorescence is activated. More
PCR = more probe cleavage = more fluorescence.
• Quantitative PCR (qPCR): amplification is detected by measuring an

increase in fluorescence; gel electrophoresis is not used in qPCR
Detection with fluorescence occurs as amplification progresses
Detection with fluorescence is more sensitive than gel electrophoresis
Detection with fluorescence is more accurate than gel electrophoresis
• To accurately measure amplification, fluorescence is measured

during the exponential phase because the fluorescence signal is
very strong during this phase
Lab Techniques
Quantitative PCR (qPCR)
• Cq values are used to quantify amplification of qPCR samples
• Cq value: cycle number that fluorescence of the sample exceeds a pre-

determined threshold. The threshold is set by experimenter to detect
fluorescence of gene in exponential phase.
• qPCR is always run with multiple samples so you can compare Cq values
Sample 1 Sample 2 • Cq values are relative to amount of template in sample

Cq = 12 Cq = 26
• Lower Cq values = higher amount of gene expression because sample

exceeds threshold at earlier cycle number
RNA Analysis Techniques
Using qPCR to measure gene expression in two different samples
Normal cells Cancer cells Normal Cancer

Cq = 12 Cq = 26
2. Isolate RNA 4. qPCR with p53 primers

Lower Cq value = higher
gene expression
3. cDNA conversion
= p53 mRNA = p53 mRNA = p53 cDNA = p53 cDNA
Normal cells have more p53 mRNA

(higher gene expression)
Lab Techniques
Southern Blot
• If two different samples of DNA have been denatured (single-
stranded) and are mixed, complementary sequences will form
hybrid duplexes
• This phenomenon is called hybridization: two complementary

single-stranded DNA (or RNA) sequences bind to form double-
stranded molecule
• Scientists can utilize hybridization with a probe: single-

stranded DNA (or RNA) with specific sequence that detects the
presence of another sequence within a sample
Radioactive probe is composed of nucleotides that contain a

32P atom which emits radiation
A probe can be radioactive or non-radioactive

Lab Techniques
Southern Blot
• Southern blot: lab technique that uses gel electrophoresis,
hybridization, and autoradiography to detect and quantify
a specific DNA sequence
Critical Steps in Southern Blot

1. Isolate genomic DNA from cells and cut DNA with
restriction enzyme
2. Use gel electrophoresis to separate DNA fragments by

dsDNA size (DNA is double-stranded)
ssDNA 3. Denature DNA and transfer from gel to membrane
4. Incubate membrane with solution that has single-

stranded radioactive DNA probe; hybridization occurs
5. Use autoradiography to detect probe

Lab Techniques
• Fluorescence in situ hybridization (FISH) is a lab technique

that allows visualization of specific DNA sequences or
entire chromosomes within a cell (no electrophoresis)
• FISH probe is made in a lab and contains nucleotides that

emit fluorescence (no radiation)
• Visualization of gene sequences on chromosomes allows

scientists to detect and diagnose genetic diseases
Lab Techniques
Critical steps in DNA FISH
1. Collect cells arrested in metaphase and fix cells to glass slide
2. Denature DNA in fixed cells to make single-stranded chromosomal DNA
3. Add probe that binds (hybridizes) to a specific DNA sequence on the

chromosomes
4. Analyze chromosomes in fixed cells with fluorescence microscope and look

for presence of probe; probe indicates presence or absence of DNA
mutation on chromosome
Chromosomes are typically blue because of DAPI: fluorescent stain that

binds to double-stranded DNA
Prokaryotic Genome Organization
Bacterial Chromosome Compaction
• To fit within the cell, the bacterial chromosome must be

very compact (1000-fold)
• Loop domains are formed by DNA-binding proteins
• Loop domains compact the DNA by 10-fold
• DNA is further compacted by twisting looped DNA to

generate DNA supercoiling
• Loop domains + supercoiling = 1000-fold compaction
• Supercoiling is regulated by proteins (topoisomerases)

Topoisomerases
DNA Supercoiling
• DNA Supercoiling occurs on all chromosomes in all cells

(prokaryotic and eukaryotic)
Topoisomerases
DNA Supercoiling
• Relaxed B-DNA has one complete turn every 10.5 bases
84 bp segment has 8 turns in relaxed state (no supercoiling)
DNA underwinding can remove one turn; double-helix has

one turn every 12 bases (not stable/strained)
• DNA underwinding: DNA strand has fewer turns than would

be expected for B-DNA; DNA underwinding generates
structural strain
Topoisomerases
DNA Supercoiling


structural strain
• Structural strain is accommodated by negative supercoiling

Topoisomerases
DNA Supercoiling


structural strain
• Structural strain is accommodated by negative supercoiling or

strand separation
• DNA underwinding promotes compaction and facilitates

strand separation
Topoisomerases
Type I and Type II Topoisomerases
Topoisomerase Class Function • Topoisomerases regulate DNA supercoiling in

Bacteria prokaryotic and eukaryotic cells by increasing
Topoisomerase I Type I Relaxes negative supercoils or decreasing turns in double helix
Topoisomerase II Type II Introduces negative supercoils
(DNA gyrase)
• All topoisomerases change DNA structure by
breaking one or both DNA strands, changing
number of turns, then ligating strands
• There are two classes of topoisomerases
Type II topoisomerases use ATP to break both

DNA strands, promote DNA underwinding,
then ligate both strands
Type I topoisomerases break one DNA strand,

relieve DNA underwinding, then ligate DNA
strand (ATP is not used)
Eukaryotic Genome Organization
• When chromatin is analyzed using SDS-PAGE, five histone proteins were

identified (H1, H3, H2B, H2A, and H4)
• H2A, H2B, H3, and H4 were present in equal amounts; H1 is present in

about half the amount compared to the other histone proteins
• H2A, H2B, H3, and H4 are core histones; two copies of each combine to
form histone octamer (nucleosome)
H1 histone is not a core histone

Eukaryotic Genome Organization
• Nucleosomes compact eukaryotic DNA six to seven-fold, but cells need

ten-thousand-fold compaction to fit DNA into nucleus
• When 147 bp of DNA is wrapped around histone octamer, 10 nm

filament forms (beads on a string); 10 nm filament doesn’t exist in cells
• Addition of H1 histone to nucleosome leads to about 168 bp wrapped

around nucleosome and alters DNA entry and exit angles
• Different entry/exit angles of DNA promotes formation of 30 nm

filament which is believed to exist in cells
• 30 nm filament compacts eukaryotic DNA 35 to 40-fold; higher level of

packaging compared to beads on a string (10 nm filament)
Histone Modifications
• Most of the histone octamer is tightly packed as globular

protein that interacts with DNA
• Each histone protein in octamer has a histone tail: N-

terminus of protein that protrudes from globular protein
• Most of the histone octamer is tightly packed as globular

protein that interacts with DNA
• Each histone protein in octamer has a histone tail: N-

terminus of protein that protrudes from globular protein
• Histone tails form intermolecular contacts with adjacent

nucleosomes that regulates chromosome compaction
• Histone modifications: chemical modifications to amino

acid side chains in N-terminal tail of histone
• Histone modifications influence molecular interactions

between nucleosomes so they can change compaction
• N-terminal tail of H3 has many important histone

modifications that regulate chromosome compaction
• For this course, focus will be on histone modifications

at H3K4, H3K9, and H3K27
• Acetylation and methylation can occur at H3K4, H3K9,

and H3K27
• Acetylation at H3K4, H3K9, and H3K27 is associated

with euchromatin and transcriptional activation
Histone acetyltransferase (HAT) adds acetyl group
lysine amino acid in N-terminal tail
Histone deacetylase (HDAC) removes acetyl group

lysine amino acid in N-terminal tail



and H3K27

• Histone methylation is more complicated

H3K4me: euchromatin/active transcription
H3K4me = methylation at 4th lysine in histone 3
Methyltransferase adds methylation to lysine

Demethylase removes methylation from lysine



and H3K27


H3K9me: heterochromatin/no transcription




and H3K27



Basics of DNA Replication
DNA Replication Requires Several Proteins
DNA helicase breaks the hydrogen bonds between DNA strands to initiate DNA replication
Topoisomerase removes supercoiling generated by helicase
Primase synthesizes RNA primers to give the DNA polymerase a 3’ OH
DNA polymerase synthesizes the new DNA strand in the 5’ to 3’ direction (needs primer)
Leading and Lagging Strands
• At the replication fork, there are two DNA strands in

opposite orientation
Replication Fork
• DNA polymerase adds nucleotides in the 5’ to 3’ direction
• Only one strand of DNA can be synthesized in the same

direction as the replication fork (leading strand)
• The leading strand is continuous
• The other strand (lagging strand) is synthesized in the

opposite direction as the replication fork
• The lagging strand is synthesized in segments called

Okazaki fragments; lagging strand is discontinuous












Origin of Replication
• Prokaryotic cells have one circular chromosome in the cytoplasm
• Prokaryotic chromosome has one origin of replication: location on

chromosome where DNA replication initiates
• Bidirectional replication: DNA is replicated in both directions at origin

of replication
Prokaryotic chromosome has two replication forks during replication

Prokaryotic chromosome has two leading strands during replication
Prokaryotic chromosome has two lagging strands during replication
• Eukaryotic chromosomes have thousands of origins of replication to

increase speed of DNA replication
• Replication at each origin is bidirectional: two replication forks, two

leading strands, and two lagging strands
Prokaryotic DNA Replication
DNA polymerases
DNA Function 3′→5′ 5′→3′ • In prokaryotic cells, DNA pol III synthesizes leading and
Polymerase Exonuclease? Exonuclease? lagging strands in 5’ to 3’ direction
Pol I Removal of RNA primers, Yes Yes
filling in gap after RNA primer • DNA pol III has 3’ to 5’ exonuclease activity
removal, DNA repair
Pol II Translesion synthesis Yes No
DNA nuclease: enzymes that degrade DNA
Pol III Chromosome replication Yes No Exonuclease: nuclease that degrades DNA from 3’ or 5’ end
Pol IV Translesion synthesis No No Endonuclease: nuclease that degrades DNA at internal location
Pol V Translesion synthesis No No
Proofreading
• In prokaryotic cells, DNA pol III synthesizes leading and
lagging strands in 5’ to 3’ direction
• DNA pol I has 5’ to 3’ and 3’ to 5’ exonuclease activity;

DNA pol III only has 3’ to 5’ exonuclease activity
• DNA pol III adds the wrong nucleotide about once

every 100,000 bases
• To reduce error rate, DNA pol III (and DNA pol I) uses
3’ to 5’ proofreading to identify and correct mistakes
1. Mismatched base causes DNA polymerase to pause
2. Pause allows mismatched base to enter pocket that cuts

DNA (exonuclease site)
3. DNA strand is digested in 3’ to 5’ direction until

mismatched base is removed
4. DNA polymerase adds correct nucleotide in 5’ to 3’

direction
Removal of RNA Primers
DNA Function 3′→5′ 5′→3′
Polymerase Exonuclease? Exonuclease?
• In prokaryotic cells, DNA pol I is used to remove RNA
Pol I Removal of RNA primers, Yes Yes
filling in gap after RNA primer
primers and replace with DNA nucleotides
removal, DNA repair
Pol II Translesion synthesis Yes No • DNA pol I is the only prokaryotic DNA polymerase with
Pol III Chromosome replication Yes No 5’ to 3’ exonuclease activity
Pol IV Translesion synthesis No No
Pol V Translesion synthesis No No

• DNA pol I is the only prokaryotic DNA polymerase with

5’ to 3’ exonuclease activity
• DNA pol I performs nick translation: concurrent 5’ to 3’

excision of RNA nucleotides and DNA polymerization

• DNA pol I is the only prokaryotic DNA polymerase with

5’ to 3’ exonuclease activity
• DNA pol I performs nick translation: concurrent 5’ to 3’

excision of RNA nucleotides and DNA polymerization
• Once all RNA is replaced with DNA, DNA ligase uses

ATP to seal “nick” in DNA backbone
DNA ligase joins two adjacent deoxyribonucleotides

already incorporated into DNA strands
Telomeres and Telomerase
Telomeres
• Telomeres: repeat sequences (6-16 nucleotides in length) that are
important for stabilizing ends of linear chromosomes
• Eukaryotic chromosomes have 3’ overhangs after DNA replication

because ends of lagging strands cannot be fully copied
Telomeres
• Telomeres: repeat sequences (6-16 nucleotides in length) that are
important for stabilizing ends of linear chromosomes
• Eukaryotic chromosomes have 3’ overhangs after DNA replication

because ends of lagging strands cannot be fully copied
• End replication problem: telomeres are shortened on both

daughter strands after each round of DNA replication
• 3’ overhangs exist on daughter strands because there is no primer

for DNA polymerase to fill in gap
Cell Senescence
Normal cells
• Cell senescence: cells stop dividing and die when telomeres

get too short
• Cell senescence is a naturally occurring process that happens

in all tissues and organs throughout lifetime of organism
• Most cells in body will undergo cell senescence after many

divisions because of telomere shortening
• Some cells (stem cells) prevent telomere shortening and do

not undergo cell senescence; stem cells are immortal
Telomerase
• To prevent telomere shortening, stem cells use telomerase
• Telomerase extends 3’ overhang by synthesizing additional

repeats using RNA as a template; RNA strand is complementary
to telomere repeat
Telomerase is composed of RNA and protein
RNA: Telomerase RNA (TR); 451 nucleotides
Protein: Telomerase Reverse Transcriptase (TERT); 1132 amino acids

Telomerase
• To prevent telomere shortening, stem cells use telomerase
• Telomerase extends 3’ overhang by synthesizing additional

repeats using telomerase RNA as a template; RNA strand is
complementary to telomere repeat
Three steps to lengthen telomeres after DNA Replication
Step one: telomerase extends 3’ overhang by synthesizing

additional repeats using telomerase RNA as a template
Step two: primase makes new RNA primer on DNA made by
telomerase
Step three: DNA polymerase makes new DNA strand
• Extension of 3’ overhang by telomerase prevents telomere

shortening after DNA replication
DNA Repair
DNA mutations are permanent changes in DNA sequences within organisms
Two main categories of DNA mutations are point mutations and chromosome mutations
Point Mutation: one base change Chromosome mutation: segment of

in the DNA sequence chromosome is lost, duplicated or moved
DNA Repair
• There are different types of base substitutions (point mutation where one base is changed)
• Silent mutation: base substitution that changes DNA sequence but does not alter the
amino acid sequence, so protein function is not altered
• Missense mutation: base substitution that changes DNA sequence which results in altering
one amino acid. Protein function may be altered if protein shape changes
• Nonsense mutation: base substitution that changes DNA sequence and generates a
premature stop codon. Protein function is altered because many amino acids are changed
DNA Repair
• Point mutations that alter protein function (cause disease) can
have two different effects:
Loss-of-function mutations: change in DNA sequence destroys

protein function; typically recessive in diploid organism
Loss-of-function mutations can be caused by changing protein

No mutation Point mutation Point mutation
Normal function changes protein decreases gene shape or decreasing gene expression
shape expression
Gain-of-function mutations: change in DNA sequence makes

protein hyperactive (rare); typically dominant in diploid organism
Gain-of-function mutations can be caused by changing protein

shape or increasing gene expression
No mutation Point mutation changes
Normal function protein shape or increases
gene expression
DNA Repair
Point mutations are caused by naturally occurring processes
(spontaneous) or environmental exposures (induced)
Spontaneous Mutations Induced Mutations

• Occurs naturally, no mutagen involved • Caused by exposure to a mutagen
(chemical, drug, radiation)
• Very low rate as long DNA repair
mechanisms are functional • Much higher rate compared to
spontaneous mutation
DNA Repair
Causes of Spontaneous Mutations
• Spontaneous mutations occur through several different

mechanisms
• Errors in DNA replication: mistakes by DNA polymerase can

generate DNA mutations if mismatched bases are not repaired by
proofreading or mismatch repair
• Oxidative damage: reactive oxygen species (ROS) made by cell

promotes oxidation of DNA bases (G to 8-oxoG)
• Depurination: covalent bond between deoxyribose sugar and

purine base (A or G) is spontaneously broken by hydrolysis
• Deamination can spontaneously remove amino group (NH2) from

base in nucleotide
DNA Repair
Causes of Induced Mutations
• Some chemicals form DNA adducts when exposed to DNA
• DNA adduct: covalent modification of base in nucleotide that is

caused by exposure to a chemical; chemical attaches to base
• DNA adducts create base substitutions by altering base pairing of

nucleotide (G with DNA adduct pairs with A instead of C during DNA
replication)
• Energy from UV light can promote formation of pyrimidine dimers:

covalent bond between two bases in same strand of DNA
• Radiation exposure can generate double-strand breaks

DNA Repair
Human cells have hundreds of thousands of mutations everyday
However, spontaneous and induced mutations are often corrected by DNA repair mechanisms
• Base excision repair (BER): repairs individual bases that are
abnormal; repairs small distortions in double helix caused by
endogenous sources
• Nucleotide excision repair (NER): repairs large distortions in

the double-helix typically involving more than one base and
caused by exogenous sources
• Mismatch repair (MMR): repairs mismatched bases

generated during DNA replication, not abnormal bases
• Homologous recombination repair (HRR): accurately repairs

double-strand breaks (DSBs), but only after DNA replication
• Nonhomologous end joining (NHEJ): repairs DSBs rapidly

and efficiently but generates mutations
Basics of Transcription
Initiation, Elongation, and Termination
Three Stages of Transcription
• Initiation: RNA polymerase binds to promoter and denatures

DNA strands to initiate transcription
• Elongation: RNA polymerase leaves promoter and synthesizes

RNA in the 5’ to 3’ direction using one DNA strand
• Termination: RNA polymerase reaches terminator sequence

and leaves template strand of DNA
Prokaryotic Transcription
• All prokaryotic (and eukaryotic) RNA polymerases are composed of

multiple polypeptides (subunits) that create functional enzyme
• Prokaryotic RNA polymerase has five subunits that form the RNA
polymerase core
• Addition of sixth subunit, sigma factor, to core creates RNA

polymerase holoenzyme
• All prokaryotic (and eukaryotic) RNA polymerases are composed of

multiple polypeptides (subunits) that create functional enzyme
• Prokaryotic RNA polymerase has five subunits that form the RNA
polymerase core
• Addition of sixth subunit, sigma factor, to core creates RNA

polymerase holoenzyme
• Different subunits in holoenzyme perform different functions
• Sigma factor binds transiently to core and is responsible for finding

promoter sequence in chromosomal DNA
Step 1. Initiation
• Six protein subunits on RNA polymerase combine to form
holoenzyme; binds loosely so it can slide down the DNA
• The sigma factor subunit on the RNA polymerase scans DNA for -35
and -10 sequences in the promoter
• -10 sequence: AT-rich region in promoter 10 bases upstream of TSS
Closed complex = DNA in RNA pol is double-stranded

• After sigma factor recognizes promoter, RNA polymerase binds
tightly (no more sliding)
Step 1. Initiation
• Six protein subunits on RNA polymerase combine to form
holoenzyme; binds loosely so it can slide down the DNA
• The sigma factor subunit on the RNA polymerase scans DNA for -35
and -10 sequences in the promoter
• -10 sequence: AT-rich region in promoter 10 bases upstream of TSS
• After sigma factor recognizes promoter, RNA polymerase binds

tightly (no more sliding)
• To begin synthesizing RNA, DNA strands are separated at the -10

sequence (AT-rich region) to create open complex
• Once RNA synthesis begins, RNA polymerase leaves promoter to

start elongation phase and sigma factor is released
Step 2. Elongation
• Initiation phase involves binding to promoter to

induce formation of open complex
Open complex: DNA is partially unwound near a

region 10bp upstream of TSS
• Transcription is initiated inside open complex

Step 2. Elongation
• Initiation phase involves binding to promoter to

induce formation of open complex
Open complex: DNA is partially unwound near a

region 10bp upstream of TSS
• Transcription is initiated inside open complex
• Elongation phase begins when RNA polymerase

has left promoter (promoter clearance)
• Elongation complex binds tightly to template DNA

strand to synthesize RNA in 5’ to 3’ direction
Step 2. Elongation
• In open complex, channels provide entry for DNA and RNTPs;

exit for synthesized RNA
• DNA strands in RNA pol core are held apart by pin: structure
that keeps transcription bubble open within enzyme
• RNA strand can form 8 base pairs with DNA template;

formation of first couple phosphodiester bonds is unstable
• Abortive initiation: binding and release of short RNA strands

from DNA at early stages of transcription
• After formation of ~10 phosphodiester bonds; elongation

complex forms and continues until termination
Transcription in Eukaryotes
RNA Polymerases
• Prokaryotic cells have one RNA polymerase that makes

three types of RNA: rRNA, tRNA, and mRNA
• Eukaryotic cells have three RNA polymerases that make

many types of RNA: rRNA, tRNA, mRNA, microRNA,
snRNA, snoRNA, piRNA, telomerase RNA
RNA Polymerases

• Eukaryotic cells have three RNA polymerase that make

• RNA polymerase I produces most of the ribosomal RNA

(rRNA) in the cell; accounts for 80% of all transcription
RNA pol I produces large rRNA strands (>7000 bases)

but produces low number
RNA Polymerases



• RNA polymerase III produces all of the tRNA and some

rRNA in the cell
RNA pol III produces small tRNA strands (<300 bases)

but produces high number
RNA Polymerases



• RNA polymerase III produces all of the tRNA and some

rRNA in the cell
• RNA polymerase II produces all of the mRNA,

microRNAs, and some noncoding RNAs
Initiation in Eukaryotes
• Five general transcription factors are used to recruit
RNA pol II to promoter
TFIID
TFIIB
TFIIF
TFIIE
TFIIH
• Five general transcription factors (GTFs) are used to
recruit RNA pol II to promoter
TFIID and TFIIH are large protein complexes with

many subunits (like RNA polymerase)
• TFIID binds to TATA box in promoter using subunit

called TATA-binding protein (TBP)
• TBP binds to TATA box near -30 position in promoter


• TBP-associated factors (TAFs) are subunits of TFIID;

recognize promoter and histone modifications
TAFs bind to initiator sequence (Inr), downstream

promoter element (DPE), histones with acetylation,
and histones with methylation
TAFs recruit TFIID to promoters that do not have a

TATA box

• TBP-associated factors (TAFs) recognize promoter

sequences and histone modifications
• TFIIB binds to TBP with C-terminal domain; binds to

RNA pol II with N-terminal domain
TFIIB binds to TFIIB recognition element (BRE)




• TFIIF is bound to RNA pol II; helps it interact with

TFIIB at promoter



• TFIIF is bound to RNA pol II; helps it interact with

TFIIB at promoter
• TFIIE and TFIIH bind last and form the preinitiation

complex: RNA pol II + five GTFs
• Preinitiation complex converts to initiation complex by
unwinding the DNA
Elongation in Eukaryotes
• Preinitiation complex converts to initiation complex by
unwinding the DNA
• TFIIH uses helicase activity to form open complex at promoter
• TFIIH uses kinase activity to phosphorylate serine residues in C-

terminus domain (CTD) of RNA polymerase II
• Phosphorylation of CTD promotes release of TFIIB, TFIIE, TFIIH

from core promoter
• Mediator brings regulatory transcription factors to promoter

so they can regulate activity of RNA polymerase II
• Mediator also regulates phosphorylation of RNA polymerase

(important for transition to elongation phase of transcription)
• RNA polymerase II will undergo abortive initiation until ~10

bases are added (similar to prokaryotic RNA polymerase)
• Mediator brings regulatory transcription factors to promoter

so they can regulate activity of RNA polymerase II
• Mediator also regulates phosphorylation of RNA polymerase

(important for transition to elongation phase of transcription)
• RNA polymerase II will undergo abortive initiation until ~10

bases are added
• At termination, RNA polymerase II is dephosphorylated and

recycled so it can transcribe another gene
RNA Processing
• Prokaryotic and eukaryotic cells make different types of
RNA to perform different functions
• Many bacterial mRNAs are translated while transcription

is occurring; no processing
• Most eukaryotic mRNAs are modified during transcription

in a process called RNA processing
• RNA polymerase II produces primary RNA transcript:

initial mRNA made from transcription; needs to be
processed before it can be used in translation
• Primary transcript undergoes 5’ capping, RNA splicing,

and 3’ polyadenylation to prepare for translation
RNA Processing
Steps for 5’ capping of primary transcript
Step one: RNA triphosphatase (RTPase) hydrolyzes phosphate

from base at 5’ end in primary transcript
Step two: Guanylyltransferase (GTase) hydrolyzes GTP and

attaches GMP to base at 5’ end with diphosphate; GTase
forms 5’, 5’ triphosphate bond
Step three: Guanylyl-7-methyltransferase attached a methyl

group to N7 on guanine base
Step four: additional methyl groups are often added to 2’

hydroxyl groups on first and second nucleotides next to cap
by 2’-O-methyltransferase
RNA Processing
• Only RNAs made by RNA pol II are capped because capping

enzyme (guanylyltransferase) associates with CTD of RNA pol II
• Guanylyltranferase dissociates from mRNA and cap-binding

complex (CBC) binds to 5’ cap to help with translation
• 5’ cap has multiple functions for mRNAs

-Protects mRNAs from degradation
-Contributes to nuclear export
-Increases efficiency of RNA splicing
-Contributes to initiation of translation

RNA Processing
• 3’ end of mRNA needs to be modified for protection

from exoribonucleases; mRNAs have 3’ polyA tail
• Polyadenylation enzymes are bound to the C-terminal

domain of RNA pol II
Steps for 3’ polyadenylation

Step one: RNA pol II transcribes polyadenylation signal
(AAUAAA)
Step two: Endonuclease cleaves RNA after signal
Step three: Polyadenylated polymerase (PAP) adds 80-250

adenines to cleaved end; no template required
Step four: PolyA binding protein (PABP) binds to

polyadenylation and protects 3’ end from degradation
RNA Processing
5’ cap is added at the beginning of transcription and the 3’ poly A tail is attached towards the end of transcription
RNA Splicing
5’ cap is added at the beginning of transcription and the poly A tail is attached towards the end of transcription
RNA splicing happens during transcription
RNA splicing: introns (and exons) are removed from primary transcript using protein complex called the spliceosome
RNA Splicing
• In eukaryotic cells, introns have to be removed before mRNA

can be used to make protein
• Spliceosome contains small nuclear ribonucleoproteins

(snRNPs): RNA-protein complexes that are critical for removing
introns from primary transcript
• snRNA within snRNPs uses base-pairing to identify sequences

(intron/exon boundaries) that are important for splicing
Spliceosome = complex of multiple snRNPs on mRNA

RNA Splicing
• Spliceosome uses three different consensus sequences for

RNA splicing: 5’ splice site, branch point, and 3’ splice site
• snRNAs in snRNPs recognize consensus sequences in intron to

indicate to spliceosome where to make two cuts
5’ splice site = 5’ end of intron always has GU
3’ splice site = 3’ end of intron always has AG
Branch point = A that is close to 3’ end

RNA Splicing
Overall Strategy of RNA Splicing
1st step: snRNPs bind to consensus sequences (5’ splice
site and branch site); forms spliceosome to position sites
2nd step, 1st reaction: 5’ splice site binds to branch site to

produce lariat
3rd Step, 2nd reaction: 3’ OH on exon 1 creates phosphodiester

bond to link exons
Alternative Splicing
(exon 3 is missing)
(exon 2 is missing)
(exons 3 and 5 are missing)
Alternative RNA splicing is a different type of RNA splicing that is used to remove or modify exons from a primary
RNA transcript to produce slightly different proteins; some exons are not critical for protein function
Alternative splicing allows for one gene to code for several different proteins
Around 70% of human genes with multiple exons undergo alternative splicing
Abnormal splice variants are commonly associated with human diseases (caused by splice site mutations)
• Cells use several types of alternative splicing to make

different proteins from one gene:
Exon skipping

Exon skipping
Intron retention

Exon skipping
A and B are different 3’ splice sites

Intron retention
Alternative 3’ splice sites


Exon skipping
Intron retention


Exon skipping
Intron retention


Exon skipping
Intron retention

• Mechanisms of alternative splicing are poorly
understood
• Splice variants have the same constitutive

exons: exons necessary for protein function
• Splice variants have the different alternative

exons: exons unnecessary for protein function
• Mistakes in RNA splicing have been associated with a

wide variety of diseases
• Splice site mutation: change in DNA sequence that

alters function of spliceosome during RNA splicing
• Splice site mutations can promote intron retention:

snRNPs do not cut out intron
• Splice site mutations can promote exon skipping:

snRNPs cut out introns and constitutive exon
RNA Editing
• Some RNAs also undergo RNA editing co-

transcriptionally and often before RNA splicing
• RNA editing: changes to the mature RNA sequence

without changing genomic DNA
• In humans, many primary transcripts are edited by

adenosine deaminase acting on RNA (ADAR): enzyme
that catalyzes conversion of adenosine to inosine
• ADAR modifies primary transcript for glutamate

receptor; changes CAG codon to CIG
Inosine base pairs with cytosine (acts like guanine)
CIG = CGG = codon for arginine
• Recent studies suggest there are over 23,000 editing

sites in the human genome
RNA Editing
• Another RNA editing enzyme used by human cells is

cytidine deaminase: coverts cytosine to uracil through
deamination
RNA Editing
• Another RNA editing enzyme used by human cells is

cytidine deaminase: coverts cytosine to uracil through
deamination
• Cytidine deaminases regulate expression of ApoB in

liver and intestinal cells
• In the liver, full length ApoB (ApoB100) is expressed;

no RNA editing in liver cells
• In the intestine, ApoB mRNA is edited which

generates premature stop codon; truncated ApoB
(ApoB48) is expressed
Introduction to Gene Regulation
Constitutive vs. Regulated Gene Expression
Constitutive Genes Regulated Genes

Genes are always expressed at the same rate Genes that are expressed in response to change
Housekeeping genes: genes that are essential for Rate of gene expression is determined by needs
life are always expressed of cell
Most genes in multicellular organisms are regulated
ON OFF ON
Examples: rRNA genes and general transcription factors Examples: genes for metabolizing different types of food
Introduction to Gene Regulation
Constitutive vs. Regulated Gene Expression
• The Human body has 50 trillion cells, and over 200

different cell types
• All cells in a human body have the same DNA sequence
• Differential gene expression: different cell types express

different genes
• Expressing different genes allows different cell types to

perform different functions
Regulatory Transcription Factors
Activators and Repressors
• Eukaryotic cells use two different types of regulatory transcription
factors: activators and repressors
• Activators bind to enhancers: regulatory sequences in DNA;

binding site for activator protein
• Activator binds to enhancer = rate of transcription is increased

Gene Regulation
Activators and Repressors
• Eukaryotic cells use two different types of regulatory transcription
factors: activators and repressors
• Activators bind to enhancers: regulatory sequences in DNA;

binding site for activator protein
• Activator binds to enhancer = rate of transcription is increased
• Repressors bind to silencers: regulatory sequences in DNA;

binding site for repressor protein
• Repressor binds to silencer = rate of transcription is decreased

Gene Regulation
Effectors
• Regulatory proteins (activators and repressors) bind to DNA

and influence transcription of genes; always present
• Effectors bind to regulatory proteins to change function;

presence of effector indicates change in environment
Gene Regulation
Glucose present, lactose absent
• Cell can use glucose for energy, does not need lac genes
to process lactose as energy source
• Repressor is bound to DNA; activator not bound to DNA

• No polycistronic mRNA for lac genes is produced
Glucose present, lactose present

• Cell can use glucose and lactose for energy, does not
need lac genes to process lactose as energy source
• Effector binds to repressor and dissociates from DNA;

activator not bound to DNA
• Some polycistronic mRNA for lac genes is produced
Gene Regulation
Glucose present, lactose absent
• Cell can use glucose for energy, does not need lac genes
to process lactose as energy source
• Repressor is bound to DNA; activator not bound to DNA

• No polycistronic mRNA for lac genes is produced
Glucose present, lactose present

• Cell can use glucose and lactose for energy, does not
need lac genes to process lactose as energy source
• Effector binds to repressor and dissociates from DNA;

activator not bound to DNA
• Some polycistronic mRNA for lac genes is produced
Glucose absent, lactose present

• Cell needs lac genes to use lactose as energy source
• Repressor not bound to DNA; different effector binds to
activator which promotes binding and Pol II recruitment
• Abundant polycistronic mRNA for lac genes is produced
Gene Regulation
Each eukaryotic genes has many regulatory binding sites because they are regulated by activators and repressors
If every gene required unique regulatory proteins, it would be too energetically costly for the cell
Eukaryotic cells use combinatorial control: genes use common regulatory proteins, but each gene uses a specific combination
of regulatory proteins to activate or inhibit expression
Gene Regulation
DNA-Binding Protein Structure
• The twist of the double helix forms two unequal
surfaces on each turn: the major and minor grooves
Gene Regulation
• The twist of the double helix forms two unequal surfaces
on each turn: the major and minor grooves
• Transcription factors interact with major groove

because there are more non-covalent interactions
Protein
Gene Regulation
• Transcription factors usually interact with major groove

• Transcription factors often have a recognition helix:

alpha helix that uses amino acid side chains to bind to a
specific DNA sequence in major groove
• Helix-loop-helix motif is protein structure found in many

transcription factors; 50 amino acids with two alpha
helices connect by string of amino acids (loop)
• Only one helix in helix-loop-helix is the recognition

helix; other helix used for dimerization
Gene Regulation
• Transcription factors usually interact with major groove

• Transcription factors often have a recognition helix:

alpha helix that uses amino acid side chains to bind to a
specific DNA sequence in major groove
• Helix-loop-helix motif is protein structure found in many

transcription factors; 50 amino acids with two alpha
helices connect by string of amino acids (loop)
• Only one helix in helix-loop-helix is the recognition

helix; other helix used for dimerization
• Regulatory sequences are often inverted repeats; many

activators and repressors bind to DNA as dimers (two
subunits bind to act as one protein)
Gene Regulation
Prokaryotic Cells Eukaryotic Cells
• Prokaryotic and eukaryotic cells have a different
transcriptional ground state: inherent activity of
promoters without regulatory mechanisms
• Prokaryotic ground state is on: RNA polymerase

can bind and initiate transcription in absence of
activators and repressors; regulatory mechanisms
Ground state: On Ground state: Off
used to inhibit gene expression
• Eukaryotic ground state is off: promoters are

inactive in absence of activators and repressors;
regulatory mechanisms used to activate expression
Primary regulation mechanism: Primary regulation mechanism:

negative regulation positive regulation
Gene Regulation
• Prokaryotic and eukaryotic cells have a different
transcriptional ground state: inherent activity of
promoters without regulatory mechanisms
• Prokaryotic ground state is on: RNA polymerase

can bind and initiate transcription in absence of
activators and repressors; regulatory mechanism
used to inhibit gene expression
• Eukaryotic ground state is off: promoters are

inactive in absence of activators and repressors;
regulatory mechanism used to activate expression
• Majority of eukaryotic genes are repressed in

ground state due to chromatin; need activators to
stimulate expression
Epigenetics
Epigenetics refers to the study of heritable changes in gene expression that occur
without alterations to the DNA sequence
• Same DNA sequence, different epigenetic modifications:
DNA Mutation chemical modifications to DNA and histones that regulate
transcription
• Epigenetic modifications and DNA mutations impact human

traits because they are heritable and alter gene expression
• If a DNA mutation is established in a cell, it is heritable because

DNA mutation is maintained during cell division
Epigenetic Modification
• If an epigenetic modification is established in a cell, it is

heritable because chemical modification is maintained during
cell division
Epigenetics
Because CpGs are symmetric there is always a CpG site on a complementary strand of DNA in the same orientation (5’ to 3’)
Maintenance methylation: DNMT localizes to replication fork during DNA replication and methylates CpG dinucleotides on
daughter strand of DNA if CpG site on template strand is methylated
Overview of Ribosomes
• Ribosome: large protein complex that synthesizes proteins
using genetic information within mRNA
• Ribosomes are essential for life; observed in all living cells
• All ribosomes have large and small subunits that perform

specific roles during translation
• When ribosomes are not actively synthesizing protein,

subunits are separated
Small subunit matches the tRNAs to codons on mRNA
Large subunit generates peptide bonds between amino

acids
Ribosome Biogenesis
• Eukaryotic cells use all three RNA polymerases to

transcribe ribosomal RNA and proteins
• RNA polymerase II makes all mRNA; all ribosomal

proteins made by RNA Pol II
• RNA polymerase I makes most rRNA; 47S/45S pre-

rRNA contains 18S, 5.8S, and 28S RNAs made by
RNA pol I
• RNA polymerase III makes some rRNA and all

tRNA; 5S rRNA made by RNA pol III
Ribosome Biogenesis
Key events in eukaryotic ribosome biogenesis
47S rRNA made in nucleolus by RNA pol I; 5S rRNA
made in nucleus by RNA pol III; ribosomal protein
mRNA made by RNA pol II in nucleus
90S ribosome forms in nucleolus: assembly factors

and snoRNAs bind to pre-RNA during transcription
Ribosomal proteins made in cytoplasm, imported into

nucleolus; 18S binds to small ribosomal proteins, 28S,
5.8S, and 5S bind to large ribosomal proteins
pre-40S and pre-60S subunits continue assembly in

nucleolus nucleus; exported to cytoplasm
In cytoplasm, 40S and 60S become functional

subunits that can perform protein synthesis
Ribosome Biogenesis
90S pre-ribosome in nucleolus contains rRNAs, sno-RNPs, assembly factors, and all ribosomal proteins
pre 40S and 60S ribosomes continue assembly in nucleolus and nucleus; start releasing assembly factors
Functional 40S and 60S subunits form in cytoplasm; finish releasing assembly factors
Translation
The Genetic Code
Genetic code: set of rules by which information encoded within genetic material (DNA or
mRNA) is translated into protein by all living cells (universal)
Three base sequence in codon with four DNA bases gives 64 codons (43 = 64)
However, there are only 20 amino acids, so the genetic code is degenerate – one amino
acid often uses several different codons
Translation
The Genetic Code
• All 64 codons are used in translation; 61 used to
code for 20 amino acids
• Genetic code is degenerate because many tRNAs

can recognize more than codon
• Wobble bases: bases in codon and anticodon that

do not always follow base-pairing rule
Translation
The Genetic Code


• tRNAs with G or U in 5’ position (wobble position)

of anticodon can recognize two codons; G/U
binding more stable between tRNA and mRNA
• tRNAs with inosine in wobble position can

recognize three codons
Translation
The Genetic Code




• Some amino acids require multiple tRNAs to

recognize all codons
Translation
The Genetic Code




Advantages of degenerate genetic code: cells require
fewer tRNAs and silent mutations • Some amino acids require multiple tRNAs to
recognize all codons
Translation
• Amino acids are attached to 3’ end of mature tRNA in cytosol by
aminoacyl-tRNA synthetase (ARS)
• Human cells make 20 different ARSs; one for each amino acid
• ARS needs to recognize specific amino acid and specific tRNA with
two different binding sites
• Amino acids are added to tRNAs in two steps
Step one: ATP and amino acid bind to ARS; ATP is used
to link amino acid to AMP forming aminoacyl-AMP
Step two: tRNA binds to ARS; amino acid is transferred

from AMP to the tRNA to form aminoacyl-tRNA, tRNA is
now charged
Translation
• Prokaryotes and eukaryotes use initiation,
elongation, and termination stages during
translation
Initiation: ribosomal subunits, mRNA, and

initiator tRNA form initiation complex at start
codon; reading frame established, initiator
tRNA is in P site, codon for next amino acid
enters the A site
Elongation: ribosome moves down mRNA

and adds amino acids to growing chain by
forming peptide bonds; charged tRNAs enter
A site and add amino acid to chain
Termination: ribosome reaches stop codon,

release factor enters A site and mRNA-
ribosome complex falls apart
Translation
Initiation
• Different tRNAs for methionine have different sequences
and base modifications
• In prokaryotic cells, initiator tRNA is created by adding a

formyl group to methionine attached to Met-tRNAfMet
Translation
Initiation

• Initiation of translation in prokaryotes requires three

initiation factors (IF-1, IF-2, and IF-3) and three steps
Translation
Initiation


Step one: Two initiation factors, IF-1 and IF-3, bind to 30S
subunit. IF-1 blocks tRNA binding, IF-3 prevents premature
activation of ribosome; mRNA binds to Shine-Dalgarno
Translation
Initiation


Step one: Two initiation factors, IF-1 and IF-3, bind to 30S
Step two: IF-2 (with GTP attached) binds to 30S subunit.

Initiator tRNA binds to start codon in P site
Translation
Initiation


Ribosome is
GAP for IF-2 Step one: Two initiation factors, IF-1 and IF-3, bind to 30S
Step two: IF-2 (with GTP attached) binds to 30S subunit.

Initiator tRNA binds to start codon in P site
Step three: GTP is hydrolyzed, 30S subunit changes, IF3

released, large subunit binds, other factors dissociate, and
initiation complex is formed
Translation
Elongation
• After the initiation complex is formed at start codon, elongation
proceeds in three repeating steps
Step one: aminoacyl-tRNA binds to A site in ribosome using base-

pairing between anticodon and codon
Step two: peptide bond is formed between amino acids in P and

A sites, growing polypeptide transferred to A site
Step three: translocation shifts tRNA without amino acid to E site,

tRNA with polypeptide to P site, new codon in A site
Translation
• Translation is similar between prokaryotic
and eukaryotic cells
• Biggest differences are in initiation stage
• Beginning of initiation is similar

Initiation factors keep ribosomal subunits
separated and prevent entry of tRNA
Translation

Initiation factors keep ribosomal subunits
separated and prevent entry of tRNA
Initiation factor (eIF2 with GTP) recruits

initiator tRNA to start codon in P site
Translation
• Eukaryotic cells have two tRNAs for Met but

no formyl group on Met in initiator tRNA
eIF2-GTP recognizes shape of initiator tRNA

and recruits it to P site in small subunit
mRNA not bound to ribosome yet
43S pre-initiation complex is now ready to

receive mRNA
Translation
• Eukaryotic cells have two tRNAs for Met; no

formyl group on Met in initiator tRNA
• 5’ cap is used to recruit mRNA to ribosome
Prokaryotic cells use Shine-Dalgarno

sequence to recruit mRNA to ribosome
Translation
eIF4F is a complex of proteins. One subunit (eIF4E) binds to 5’ cap. Another
subunit (eIF4G) connects complex to small ribosomal unit (binds to IF3). A • Biggest differences are in initiation stage
third subunit (eIF4A) hydrolyzes ATP to promote attachment to ribosome.
eIF4F = eIF4E + eIF4G + eIF4A • Eukaryotic cells have two tRNAs for Met; no

Initiation factor (eIF4F) binds to 5’ cap
Translation


Initiation factor (eIF4F) binds to 5’ cap; uses
ATP to recruit mRNA to preinitiation complex
to form 48S particle
Translation


Initiation factor (eIF4F) binds to 5’ cap; uses
ATP to recruit mRNA to preinitiation complex
to form 48S particle
Complex scans mRNA in 5’ to 3’ direction to
find start codon (AUG) after 5’ cap
Translation


Initiation factor (eIF4F) binds to 5’ cap and
recruits it to preinitiation complex to form
48S particle
Complex scans mRNA in 5’ to 3’ direction to
find start codon (AUG) after 5’ cap
Base pairing between initiator tRNA and
start codon near 5’ end establishes reading
frame
Translation

• When small subunit and initiator tRNA are

bound at start codon, large subunit joins
Non-Coding RNA (ncRNA)
Typical human cell: • The importance of ncRNAs can be illustrated by the amount of ncRNA
~16,000 genes expressed
in a typical human cell
20% Protein-
• Although ~16,000 genes are expressed in a human cell, only 20% of
80% non-coding encoding genes these genes encode for proteins
genes (ncRNA) (mRNA)
• ncRNA accounts for ~80% of genes expressed in a human cell
• ncRNAs are very diverse in size and function
• Long non-coding RNAs (lncRNAs) are longer than 200 nucleotides and
are often used to localize proteins to specific locations on DNA
Typical human cell: • The importance of ncRNAs can be illustrated by the amount of ncRNA
~16,000 genes expressed
20% Protein-
• Although ~16,000 genes are expressed in a human cell, only 20% of
80% non-coding encoding genes these genes encode for proteins
genes (ncRNA) (mRNA)
• Small non-coding RNAs (e.g., microRNA) are shorter than 200

nucleotides and typically bind to mRNA to influence gene expression
• The importance of ncRNAs can be illustrated by the amount of ncRNA
Eukaryotic cells use housekeeping ncRNAs
and regulatory ncRNAs • Although ~16,000 genes are expressed in a human cell, only 20% of
these genes encode for proteins
• Small non-coding RNAs (e.g., microRNA) are shorter than 200

nucleotides and typically bind to mRNA to influence gene expression
Long Non-Coding RNA: Xist in X-inactivation
Patch of cells using the Patch of cells using the

maternal X chromosome paternal X chromosome
• X-inactivation (XCI): one of the two X chromosomes in a female mammal is inactivated and becomes condensed
• Inactive X chromosome is a Barr Body: heterochromatic, very condense structure
• X-inactivation occurs early in development and is random in the female body which produces somatic mosaicism:
some cells express the paternal X chromosome, and some cells express the maternal X chromosome
Patch of cells using the Patch of cells using the

maternal X chromosome paternal X chromosome
• X-inactivation (XCI): one of the two X chromosomes in a female mammal is inactivated and becomes condensed
• Inactive X chromosome is a Barr Body: heterochromatic, very condense structure
• X-inactivation occurs early in development and is random in the female body which produces somatic mosaicism:
some cells express the paternal X chromosome, and some cells express the maternal X chromosome
• X-inactivation causes spotted coat on calico cat (almost always female). Orange fur = active maternal X
chromosome, black fur = active paternal X chromosome
• Xist is a long non-coding RNA that is only expressed from the

inactive X chromosome (Xi) and covers the entire chromosome
• Xist expression promotes different histone modifications on Xi

and active X chromosome (Xa)
• Xist is a long non-coding RNA that is only expressed from the

inactive X chromosome (Xi) and covers the entire chromosome
• Xist expression promotes different histone modifications on Xi

and active X chromosome (Xa)
• Inactive X chromosome (Xi) has histone modifications (H3K9me

and H3K27me) that promote heterochromatin
• Active X chromosome (Xa) has histone modifications (acetylation

and H3K4me) that promote euchromatin
• X-inactivation is an epigenetic process that is critical for

mammalian development
X-inactivation is initiated early in development
X-inactivation controls gene expression without changing

the DNA sequence
X-inactivation is heritable (maintained during cell division)
• Some genes on X chromosome can escape X-inactivation
• Escaping X inactivation is not random; same genes in loops

that extend from compact domain
MicroRNA (miRNA)
• Primary miRNA (pri-miRNA) is transcribed by RNA pol II and forms
hairpin structure (several hundred nucleotides long)
• Pri-miRNA is cut by Drosha in nucleus to form precursor miRNA

(pre-miRNA) that is 70 nucleotides long and double stranded
• Pre-miRNA is exported out of nucleus and into cytosol. Dicer cuts

pre-miRNA in cytosol to make double-stranded intermediate
• Double-stranded intermediate associates with RNA-induced

silencing complex (RISC) proteins which degrade one RNA strand
• RISC is composed of single-stranded miRNA (22 nucleotides) and

RISC proteins; proteins help miRNA bind to mRNA at 3’UTR
Perfect match between miRNA and mRNA = mRNA is degraded
Partial match between miRNA and mRNA = mRNA is not translated
Partial match allows one miRNA to regulate multiple mRNAs

Short Interfering RNA (siRNA)
• Short-interfering RNA (siRNA): double-stranded RNA that
induces RNA degradation using RNAi pathway
Not transcribed from human genome; not cut by Drosha in

nucleus of human cells
Cut by Dicer in cytoplasm
Single-stranded RNA forms complex with RISC
RISC + siRNA bind to target sequence to promote RNA

degradation
CRISPR RNA (crRNA)
Transgenic Techniques
• Transgenesis: process of transferring a gene (transgene)
from one organism to another
• Bacterial transformation is a type of transgenesis; useful in

research to study DNA segments/genes
• Transgenesis: process of transferring a gene (transgene)
from one organism to another
• Bacterial transformation is a type of transgenesis; useful in

research to study DNA segments/genes
• Bacterial transgenesis can be used to make large amounts of

human proteins like insulin and human growth hormone
• Shuttle vectors are plasmids that can propagate in two
different host species
• Many shuttle vectors are used to transform one plasmid

in bacterial and yeast cells
• Various sequences are required for shuttle vectors:
Cloning site for DNA cloning: has restriction sequences
Antibiotic resistance and bacterial ori for bacterial

transformation: make copies of recombinant plasmid
Yeast ori, centromere sequence and selectable marker

used for yeast transformation
Selectable marker = gene for leucine biosynthesis

• Shuttle vectors are plasmids that can propagate in two
different host species
• Many shuttle vectors are used to transform one plasmid

in bacterial and yeast cells
• Various sequences are required for shuttle vectors:
Cloning site of DNA cloning: has restriction sites
Antibiotic resistance and bacterial ori for bacterial

transformation: make copies of recombinant plasmid
Isolate plasmid from bacteria, transform into yeast
Yeast ori, centromere sequence and selectable marker

used for yeast transformation
Selectable marker = gene for leucine biosynthesis
• Selectable marker is used to select for yeast cells that

have received plasmid during transformation
Genetically Modified Organisms (GMOs)
• Transgenic plant: plant that has been genetically modified to
create a new trait (same thing as genetically modified plant)
• To create transgenic plants, scientists use bacteria that naturally

infect plant cells and insert DNA from a plasmid
• These plant-infecting bacteria contain a plasmid (Ti plasmid) that

transfers DNA (T DNA) to the plant genome upon infection
Bacteria is called A. tumefaciens; this species is used because it

infects plant cells and naturally has a Ti plasmid


• Scientists modified Ti plasmid for DNA cloning: vector has

antibiotic resistance and cloning site in T DNA
T-DNA vector = modified Ti plasmid used for DNA cloning
Kanamycin resistance can be used to select for transformed

bacterial and plant cells


• Scientists modified Ti plasmid for DNA cloning: vector has

antibiotic resistance and cloning site in T DNA
• T-DNA vector is transformed into bacteria; transformed cells have

recombinant T-DNA vector (plasmid with cloned gene)
• Bacteria (with plasmid) infect plant cells; T DNA (with gene of

interest) is inserted into plant genome
• Transformed plant cells used to make entire plant; all plant cells
have cloned gene from T DNA
• Gene therapy is an experimental technique that can introduce

cloned genes into human cells to treat or prevent disease
• Viruses are often used to insert cloned genes into human

chromosomes to treat autosomal recessive diseases
• Viral vectors are modified viruses (cannot reproduce) that deliver

genes into human cells to restore function of cells causing disease
• Viral vectors typically deliver one cloned gene into chromosome
• Gene therapy is used to treat diseases that are caused by one non-
functional gene (monogenic disease)
• The CRISPR/Cas9 system is used to target a nuclease to a
specific location and promote gene editing
• The CRISPR/Cas9 system has two critical components: guide

RNA (gRNA) and Cas9
gRNA: single-stranded RNA that is complementary to a gene
(crRNA) and forms a secondary structure (tracrRNA)
Cas9: RNA-guided nuclease that creates DSBs
• The type of gene editing that occurs depends on the presence

or absence of donor DNA
Donor DNA present in cell: homologous recombination fixes DSB
created by Cas9 and gene replacement occurs
Donor DNA absent in cell: non-homologous end joining fixes DSB

created by Cas9 and a deletion mutation typically occurs
Human Transgenesis
• The CRISPR/Cas9 system is used to target a nuclease to a
specific location and promote gene editing
• The CRISPR/Cas9 system has two critical components: guide

RNA (gRNA) and Cas9
gRNA: single-stranded RNA that is complementary to a gene
(crRNA) and forms a secondary structure (tracrRNA)
Cas9: RNA-guided nuclease that creates DSBs
• The type of gene editing that occurs depends on the presence

or absence of donor DNA
Donor DNA present in cell: homologous recombination fixes DSB
created by Cas9, and gene replacement occurs
Donor DNA absent in cell: non-homologous end joining fixes DSB

created by Cas9, and a deletion mutation typically occurs
• CRISPR/Cas9 system uses modified components of CRISPR Cas

system in prokaryotic that destroys viral DNA
DNA Sequencing
• DNA sequencing reaction has two types of nucleotides: dNTPs
and ddNTPs
• DNA strand grows in 5’ to 3’ direction by adding dNTPs to 3’ OH

of nucleotide that is already incorporated into DNA
• Low number of ddNTPs that lack a 3’ OH are mixed with high

number of dNTPs in sequencing reaction
• When ddNTP is added to growing DNA strand, synthesis stops

because there is no 3’ OH for DNA polymerase to add nucleotide
Key steps in DNA sequencing:

1. Isolate template strand of DNA, make new strands of DNA at different
lengths, and label end of each fragment with fluorescent nucleotide
2. Fluorescent-labeled fragments are sorted by size using gel electrophoresis
3. Detector reads fluorescent labels; computer generates sequence of bases in

5’ to 3’ direction (matches fluorescence color to base)
Genomics
• Average size of human gene is 5,000 bases; can sequence 750
bases with each reaction
• Genome: all the genes and non-coding DNA in an organism; all

DNA sequences on all chromosomes
• Sequencing an individual gene is easy, sequencing an entire

genome is very difficult
Challenges to sequencing a genome:
• Organization: chromosomes are tangled together in nucleus like

a ball of yarn; cannot sequence one chromosome at a time
• Size: human genome has ~25,000 genes and 3 billion bases,

Sanger sequencing can only sequence ~750 bases at a time
Genomics
Constructing the first human genome sequence
Step one: isolate genomic DNA and cut into large fragments
(~100,000 bases) called contigs: a long continuous stretch of
chromosomal DNA that overlaps with other contigs
Step two: each contig was broken up into smaller pieces and
cloned into bacterial artificial chromosomes (BAC)
Step three: smaller pieces are sequenced (750 bases at a time);

STSs and ESTs used to assemble sequence of contig
STS = short segment of DNA that occurs once in genome
EST = STS derived from cDNA (occurs in gene sequence)
Genomics
Constructing the first human genome sequence
Step one: isolate genomic DNA and cut into large fragments
(~100,000 bases) called contigs: a long continuous stretch of
chromosomal DNA that overlaps with other contigs
Step two: each contig was broken up into smaller pieces and
cloned into bacterial artificial chromosomes (BAC)
Step three: smaller pieces are sequenced (750 bases at a time);

STSs and EST used to assemble sequence of contig
Step four: use overlapping sequences at ends of contigs to

assemble sequences of all chromosomes (reference sequence)
Genome sequence produced by Human Genome Project was

high quality, but it took too long and was too expensive
High cost and large amount of time in project was due to use
of gel during DNA sequencing
Genomics
• Genome sequence produced by Human Genome Project was
• High cost and large amount of time in project was due to use
NGS sequencing chip has
millions of small wells • Next generation sequencing(NGS): sequence millions of DNA
fragments simultaneously without using a gel
Step one: isolate genomic DNA and cut into small fragments
(50 -100 bases); adaptor sequences attached to fragments
Step two: attach small fragments (~200 million) to chip
Individual fragment is attached to one well using adaptor.

Fragment is copied within each well.
Genomics
• Next generation sequencing(NGS): sequence millions of DNA

(50 -100 bases); adaptor sequences attached to fragment
Step three: use reversible terminator sequencing to

sequence 200 million fragments simultaneously; DNA is
sequenced as new strand is being made (no gel)
Sequencing primer binds to adaptor sequence

Genomics
Nucleotides are reversible terminators (not ddNTPs)

Bind to template and emit fluorescence • High cost and large amount of time in project was due to use
Fluorescence removed of gel during DNA sequencing
Blocking group on 3’ end of nucleotide removed
Genomics
ddNTPs vs Reversible Terminators
Genomics

(50 -100 bases); adaptor sequences attached to fragment
Step three: use reversible terminator sequencing to

sequence 200 million fragments simultaneously; DNA
sequenced as new strand is being made (no gel)
Step four: computer assembles genome sequence using

sequences from DNA fragments and reference sequence
Genomics
• Genetic testing can guide treatment options for cancer
patients and identify germline mutations in families with
history of cancer; germline mutation = inherited mutation
• Germline testing: identifies germline mutations associated

with hereditary cancer; germline mutation is in every cell
• Tumor testing: identifies mutations in tumor cells; useful for

personalized medicine (identify drug specific to patient)
Germline Testing Tumor Testing
Performed on blood/saliva Performed on tumor cells
Patient may be unaffected Patient has cancer
Determine risk of Determine prognosis and

hereditary cancer identify treatment options
Genomic Imprinting
Genomic imprinting: certain genes are expressed from only one chromosome in a parent-of-origin specific manner
Paternal OFF Gene 1 ON Gene 2 OFF Gene 3

Chromosome
Unmethylated CpG
Methylated CpG
Maternal
OFF Gene 1 OFF Gene 2 ON Gene 3
Chromosome
Non-imprinted gene: gene is expressed Paternally-Expressed Maternally-Expressed

or repressed on both chromosomes Imprinted Gene Imprinted Gene
Genomic imprinting is rare, most genes are expressed from both paternal and maternal chromosomes
For most genes, if gene is turned off, then expression is repressed on both chromosomes
Eukaryotic cells can inhibit gene expression by methylating CpG sites in a CpG island in or around the promoter
With imprinted genes (genes expressed from only one chromosome) one chromosome is methylated and the
other chromosome is unmethylated; imprinted genes are expressed form paternal or maternal chromosome
Genomic Imprinting
Imprinted Genes in Mouse Genome
This is an imprinting map: diagram that shows all

imprinted genes in genome (all chromosomes)
Important characteristics of imprinted genes
1. Imprinted genes are rare; only ~100

imprinted genes in human genome on
different chromosomes
2. Imprinted genes are often clustered;

brackets on map represent clusters of
imprinted genes
Genomic Imprinting
A Typical Imprinting Cluster
Epimutation: change in DNA methylation
Paternal OFF ICR ON

Chromosome
Unmethylated CpG
Methylated CpG
Maternal
Chromosome
OFF ICR ON
Imprinted Genes
• Clustering allows multiple imprinted genes to be regulated by one imprinting control region (ICR): regulatory sequence with
a large amount of CpG sites (CpG island)
• ICRs are methylated on one chromosome; differential methylation at ICR allows expression from one chromosome
• Epimutation: change in DNA methylation that causes a change in gene expression; epimutations at ICRs alter expression of
imprinted genes and cause developmental defects
• Epimutations are often caused by environmental exposures; no change in DNA sequence is required for an epimutation
Genomic Imprinting
H19 and Igf2 Imprinting Cluster on Human Chromosome
Epimutation
11
BWS caused by epimutation at

H19/Igf2 imprinting cluster
• The H19/Igf2 imprinting cluster has two imprinted genes (H19 and Igf2) and is regulated by an ICR that is methylated on the
paternal chromosome
• H19 is expressed from the maternal chromosome, and Igf2 is expressed from the paternal chromosome
• Igf2 is important for regulating size of tissues/organs; organism must express right amount of Igf2 to have correct size
• Epimutation that promotes expression of Igf2 from both chromosomes causes Beckwith-Wiedemann Syndrome (BWS) in
humans (an overgrowth syndrome)
Epigenetic Reprogramming
• All diploid organisms use meiosis to create haploid gametes (each

gamete has one copy of every chromosome)
• Haploid gametes fuse during fertilization to make a diploid zygote (two

copies for every chromosome); zygote = one cell
• Zygote undergoes multiple rounds of mitosis to create early embryo

(compact ball of diploid cells)
• Early embryo has somatic cells and germ-line cells
• Imprinted genes are reprogrammed in germ-line cells of early embryo

Somatic cells (diploid): produce other diploid cells for organs/tissues with mitosis
Germ-line cells (diploid): produce haploid gametes (egg/sperm cells) with meiosis
• Epigenetic reprogramming: genome-wide erasure of
Diploid Zygote epigenetic marks, such as DNA methylation, during
different stages of mammalian development
• During formation of gametes, DNA methylation at ICRs

Diploid Germ-line Cell
undergoes epigenetic reprogramming
Meiosis • H19/Igf2 ICR in sperm: DNA methylation is erased, then

established on both chromosomes
Haploid Gamete
epigenetic marks, such as DNA methylation, during

• H19/Igf2 ICR in sperm: DNA methylation is erased, then

• H19/Igf2 ICR in oocyte: DNA methylation is erased, stays

erased on both chromosomes
epigenetic marks, such as DNA methylation, during

• H19/Igf2 ICR in sperm: DNA methylation is erased, then

• H19/Igf2 ICR in oocyte: DNA methylation is erased, stays

erased on both chromosomes
• Fertilization produces diploid zygote with methylated

paternal chromosome and unmethylated maternal
chromosome at H19/Igf2 ICR
• In mammalian development, epigenetic reprogramming
occurs in two phases: gametogenesis and preimplantation
development
• Gametogenesis: production of gametes; initiated in germ-line

cells of early embryo, completed in adulthood
• Preimplantation development: fertilized zygote develops into

blastocyst while migrating in fallopian tube; takes about 7
days in human development
• Gametogenesis epigenetic reprogramming: DNA methylation

erased at all imprinted and non-imprinted genes during
early stages, re-established in late stages
• In mammalian development, epigenetic reprogramming
occurs in two phases: gametogenesis and preimplantation
development
• Gametogenesis: production of gametes; initiated in germ-line

cells of early embryo, completed in adulthood
• Preimplantation development: fertilized zygote develops into

blastocyst while migrating in fallopian tube; takes about 7
days in human development
• Gametogenesis epigenetic reprogramming: DNA methylation

erased at all imprinted and non-imprinted genes during early
stages, re-established in late stages
• Preimplantation epigenetic reprogramming: DNA erased

only at non-imprinted genes; methylation at imprinted
genes in maintained
Epigenetics and IVF
• Assisted reproductive technologies (ARTs) are fertility-
related treatments in which eggs, sperm, or embryos are
manipulated
• In vitro fertilization (IVF) is the most common ART

Superovulation
• IVF requires three separate medical procedures

superovulation, embryo culture, and embryo transfer
Embryo • Superovulation: inject hormones to stimulate ovulation of

transfer multiple eggs; eggs are collected
Embryo culture
• Embryo culture: collected eggs are fertilized with sperm
and cultured for 5-7 days in lab
• Embryo transfer: highest quality embryo(s) transferred into

uterus of recipient female
Epigenetics and IVF
• Developmental Origins of Health and Disease: theory that

environmental exposures during in utero development influence fetal
growth and can predispose an individual to adult metabolic diseases
(e.g., coronary heart disease, hypertension, type II diabetes)
• Theory was founded on observations from Dutch Famine Study
Acute famine in Netherlands during World War II
Nazis imposed an embargo from October 1944 - May 1945 on all

incoming transport, including food
All Dutch citizens had daily ration of 580 - 1200 calories during
embargo; may people died of starvation
Dutch government kept very accurate pregnancy and birth records

during embargo; scientists used information to track long-term health
Epigenetics and IVF
• Researchers assessed the long-term health of individuals that
were born before, during, and after the famine
• Observation from one study: exposure to the famine during early

stages of development results in higher rates of adult obesity
• Conclusion: early stages of development are sensitive to

environmental exposures and impact long-term health
Epigenetics and IVF
• In developed countries, IVF accounts for 1-4% of total births;
vast majority of IVF children are completely healthy
• Children conceived by IVF exhibit a higher incidence of

imprinting disorders and low birth weight
Embryo Embryo
• Animal studies have demonstrated that different aspects of
Superovulation Culture Transfer the ART can induce epimutations at imprinted genes
• Medical procedures performed during an IVF cycle occur

during the early stages of development
• Medical procedures coincide with epigenetic reprogramming
Superovulation is performed when epigenetic reprogramming

occurs in gametes
Embryo culture and transfer are performed when epigenetic

reprogramming occurs in preimplantation embryo

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MBS 344: Molecular Biology

Final Exam Lecture Slides

• Don’t have to clone DNA into a plasmid to amplify a

• PCR can amplify any region of the genome using isolated

• Primers specify the region of amplification by providing

• PCR can amplify specific regions from very small

PCR requires three steps to amplify specific region of DNA:

1. Denature (95°C):high temperature separates DNA strands by

2. Anneal (~55°C):lower temperature allows primers to anneal

3. Extend (~70°C): optimal temperature for DNA polymerase to

• Agarose gel electrophoresis sorts DNA segments by size; often used to

• M = molecular weight marker (used to measure size of bands in gel);

• Should see one band in gel if PCR was successful

• Gel electrophoresis is used to visual bands and estimate size of bands

SYBR green: fluorescence molecule that binds to double-stranded

• Quantitative PCR (qPCR): amplification is detected by measuring an

• To accurately measure amplification, fluorescence is measured

• Cq values are used to quantify amplification of qPCR samples

• Cq value: cycle number that fluorescence of the sample exceeds a pre-

Sample 1 Sample 2 • Cq values are relative to amount of template in sample

• Lower Cq values = higher amount of gene expression because sample

Normal cells Cancer cells Normal Cancer

2. Isolate RNA 4. qPCR with p53 primers

= p53 mRNA = p53 mRNA = p53 cDNA = p53 cDNA

Normal cells have more p53 mRNA

• This phenomenon is called hybridization: two complementary

• Scientists can utilize hybridization with a probe: single-

Radioactive probe is composed of nucleotides that contain a

A probe can be radioactive or non-radioactive

Critical Steps in Southern Blot

2. Use gel electrophoresis to separate DNA fragments by

ssDNA 3. Denature DNA and transfer from gel to membrane

4. Incubate membrane with solution that has single-

5. Use autoradiography to detect probe

• Fluorescence in situ hybridization (FISH) is a lab technique

• FISH probe is made in a lab and contains nucleotides that

• Visualization of gene sequences on chromosomes allows

2. Denature DNA in fixed cells to make single-stranded chromosomal DNA

3. Add probe that binds (hybridizes) to a specific DNA sequence on the

4. Analyze chromosomes in fixed cells with fluorescence microscope and look

Chromosomes are typically blue because of DAPI: fluorescent stain that

• To fit within the cell, the bacterial chromosome must be

• Loop domains are formed by DNA-binding proteins

• Loop domains compact the DNA by 10-fold

• DNA is further compacted by twisting looped DNA to

• Loop domains + supercoiling = 1000-fold compaction

• Supercoiling is regulated by proteins (topoisomerases)

• DNA Supercoiling occurs on all chromosomes in all cells

• Relaxed B-DNA has one complete turn every 10.5 bases

84 bp segment has 8 turns in relaxed state (no supercoiling)

DNA underwinding can remove one turn; double-helix has

• DNA underwinding: DNA strand has fewer turns than would

• Relaxed B-DNA has one complete turn every 10.5 bases

84 bp segment has 8 turns in relaxed state (no supercoiling)

DNA underwinding can remove one turn; double-helix has

• DNA underwinding: DNA strand has fewer turns than would

• Structural strain is accommodated by negative supercoiling

• Relaxed B-DNA has one complete turn every 10.5 bases

84 bp segment has 8 turns in relaxed state (no supercoiling)

DNA underwinding can remove one turn; double-helix has