Polymerase Chain Reaction

 All cells have remarkable ability to synthesize DNA with

specific sequences
 However, inability to synthetically create DNA hampered
investigations of specific genes as we needed to start from
genomic DNA to isolate the gene sequence of interest
 Enter…PCR!
o Developed by Kary Mullis in 1983
 Awarded Nobel Prize in 1993
 PCR is…
o A chain reaction is a sequence of reactions where a
reactive product or by-product causes additional
reactions to take place
o In a chain reaction, positive feedback leads to a self-
amplifying sequence of events
 PCR is…
o Identical DNA molecules of specific nucleotide
sequences produced in vitro
o A repetitive process of 3 steps constituting 1 cycle
 Denaturation
 dsDNA is heated briefly to separate 2
strands
 Priming
 The DNA is exposed to a large excess of a pair of specific primers
– designed to bracket the region of DNA to be amplified—and the
sample is cooled to allow the primers to hybridize to
complementary sequences in the two DNA strands
 Extension
 This mixture is incubated with DNA polymerase and the four
deoxyribonucleoside triphosphates so that DNA can be
synthesized, starting from the two primers
o Automated with a thermocycler
 Most polymerase chain reaction (PCR) methods rely on thermal cycling to permit
different temperature-dependent reactions – specifically, DNA melting and enzyme-
driven DNA replication to proceed
 DNA amplification via PCR is an exponential process
o # of molecules generated after n cycles = 2n
o 30 cycles results in 230 molecules
o That’s a lot of fucking molecules
 We need to know certain things about the sequence being amplified during PCR
o Known DNA/RNA sequence
o Known homologous sequence
o Known protein sequence
 We need to have…
o A source of target DNA
 i.e. a sequence
o Set of oligonucleotide primers
 20 to 40 bp, ssDNA, chemically synthesized
 each (antiparallel) primer is complimentary to the 3’ end of the target
strand (we need to know this DNA sequence)
 Unique to target
o Enzyme capable of forming 3’ to 5’ phosphodiester bonds (DNAP)
 PCR reaction profile

 Thermally stable
DNAP
o Taq is the polymerase enzyme used in PCR
o Originally isolated from Thermus aquaticus
 Alu: a short (~300bp) repeated DNA element (SINE) found throughout genome
 tPA 25 is an Alu element found in an intron of a tPA gene (tissue plasminogen activator,
encodes a serine protease)
 Present in only some people, acts as a neutral phenotypic marker
o Useful in demonstrating PCR detection of polymorphism
 PCR reaction require many necessary components
1. Template DNA
2. Oligonucleotide primer
3. Nucleotides
4. Enzyme cofactor (Mg2+)
5. Thermally stable DNAP (Taq)
6. Buffer (Tris)
 PCR only demonstrates target presence
 Controls are important
o Negative = no template
o Positive = known template
 PCR considerations
o Specificity: how well primers target specific sequences
o Fidelity: how accurately DNA is replicated (mismatch frequency)
 Optimization is empirical!
DNA Replication
 Replication is a cyclic process
o DNA replication  transcription  translation
o DNA  RNA  Protein
o DS nucleic acid  SS nucleic acid  amino acid
 DNA replication has a semi conservative mechanism
o When genomic DNA is synthesized, each strand of a DNA double helix serves as
the template or the synthesis of a complementary strand
o DNA replication can also be dispersive in which parent strand DNA is randomly
dispersed in both daughter strands
 DNA replication synthesis is from the 5’ to 3’
o Both parental strands of the original DNA molecule serve as the template strand
o Synthesis of daughter strand occurs 5’ to 3’, and vice versa for reading of
template strand
o ***Two template strands are antiparallel, so synthesis of daughter strands is
ALSO antiparallel!***
 Bidirectional
 Addition of dNTPs occur sequentially
 o Daughter strands are synthesized by the sequential addition of dNTPs to the free
3’ OH with the release of PPi
 Release and hydrolysis of PPi creates a favorable free energy change,
driving the synthesis
o The parental DNA strand is the template for the daughter strand. It is also a
substrate in the polymerization reaction and determines the which nucleotide is
incorporated into the daughter strand via the complimentary base-pairing rules

Replication Fork
 Replication fork is a localized region of replication that moves along parental dsDNA
o Called a fork because of a Y shaped structure (think steak fork)
 Because synthesis if daughter strands is bidirectional, there must be some way for DNAP
to synthesize both ways
o However, DNAP at replication forks can only synthesize in the 5’ to 3’ direction
 Transient pieces of DNA, known as Okazaki fragments, exist at the replication fork
o Polymerized in 5’ to 3’ direction and joined after synthesis to create long DNA
chains
 Replication fork has asymmetric structure
o One DNA template strand is oriented 3’ to 5’ so the corresponding daughter
strand is synthesized 5’ to 3’
 This strand is synthesized continuously
o The DNA daughter strand that is synthesized continuously is known as the
leading strand
o Synthesis precedes that of the discontinuously synthesized daughter strand
 o The discontinuously synthesized daughter strand is built to correspond with the
template strand that runs 5’ to 3’, so it must be synthesized 3’ to 5’
 Known as the lagging strand
 Generated in short segments of DNA known as Okazaki Fragments
 Short segments are subsequently joined by ligase
o Lagging strand has an opposite direction of polymerization to the overall direction
of DNA growth
o Lagging strand is synthesized via a back stitching mechanism
 Requires only DNAP that goes 5’ to 3’

Proofreading Mechanisms
 High fidelity of DNA replication means it depends on numerous proofreading
mechanisms as well as the initial base pairing
o Mechanisms act sequentially to prevent errors in mispairing of bases
 Each nucleotide is checked
 DNAP performs the first proofreading step
o First step performed before a new nucleotide is covalently added to the growing
chain
o Correct nucleotide has higher biochemical affinity for the growing strain than an
incorrect nucleotide due to a more favorable free energy change
o DNAP can also double check the correctness of the base pairing prior to the
addition of the nucleotide due to a conformational change
o Incorrect base pairs are harder to add and diffuse away
 Second proofreading step is known as exonucleolytic proofreading
o Occurs immediately after an incorrect base pairing is catalyzed
 Very rare chance
o DNAP enzymes are very discriminatory in which DNA chains they can elongate
 Requires a previously catalyzed, base paired 3’ OH end of a primer strand
o These errors are corrected by a separate catalytic site
 Known as a 3’ to 5’ proofreading exonuclease (ε subunit)
o 3’ to 5’ proofreading exonuclease clips off any unpaired or mismatched residues
at the primer terminus
 Continues until correct base paired 3’ OH can be generated
 o DNAP can be thought of as a self – correcting enzyme that removes its own
polymerization errors as it moves along the DNA
 Because nucleotides are functionally asymmetrical, proofreading cannot occur during 3’
to 5’ synthesis
o This would remove Okazaki fragments
o No energy source  no cleavage of high energy bond to release energy
 Proofreading is essential to biological life (wow thanks Espie, no fucking shit)
DNA Polymerase III (3)
 DNA polymerase III is the primary enzyme responsible for prokaryotic DNA replication
o Composed of numerous subunits
o Very complex enzyme
 Each subunit carries out distinctive functions in the DNA replication process
 α subunit: catalyzes 5’ to 3’ phosphodiester bonds
o Big boi
o Core enzyme
 ε subunit: catalyzes a variety of biochemical reactions; phosphatase, can cleave
phosphate bonds
o Exonuclease: protein binds to end of a DNA molecule, has ability to cleave off
only terminal nucleotide
o Has 3’ to 5’ exonuclease activity
o Important for proofreading
o Core enzyme
 θ subunit: regulates and controls activity of ε subunit
o Core enzyme
 χ subunit: binds to single stranded binding proteins
 β subunit: the sliding clamp which helps the nucleotide maintain itself in the DNA base
molecule
o Stabilizes DNA polymerase on DNA substrate
o Dimeric structure w/ 2 halves that come together in its final conformation
 DNA Helicase: unwinds double helix into two single stranded DNA molecules
 γ complex: composed of one γ subunit, one δ subunit, one δ’ subunit, one χ subunit, and
one ψ subunit
o gamma subunit binds ATP
o delta subunit binds onto beta subunit
o delta prime subunit binds to gamma and delta subunits
o gamma complex catalyzes ATP in order to chaperone two beta subunits to bind to
DNA
  DNA has five polymerases
o Only DNAP1/2/3 are sufficient to maintain replication
o Each polymerase is a multimeric complex, about 1M Daltons
o DNAP1 is important for DNA repair
o DNAP1 has 3 biological activities

Okazaki Fragments
 Okazaki fragments are segments of DNA synthesized on the lagging strand
 Varying length
o Prokaryotes: ~1000 nucleotides
o Eukaryotes: 100 – 200 nucleotides
 Because one daughter strand is synthesized 5’ to 3’, the other daughter strand must be
synthesized 3’ to 5’ by obligation
o Results in necessity to discontinuously synthesize DNA in Okazaki Fragments
 Okazaki Fragments are initially RNA – DNA hybrids
RNA Primers
  DNA polymerase requires a double stranded
nucleic acid for binding
 Prior to DNA polymerization, an RNA
primer is required
o RNA that initiates DNA synthesis
o Requires as no known DNA
polymerase can synthesize
polynucleotide chains from existing
3’ OH
 RNA primer contains a properly base-paired
nucleotide with a 3ʹ-OH group at one end
o Can be elongated by the DNA
polymerase at this end
o Begins an Okazaki fragment
 Different daughter strands require varying
primers
o Leading strand requires one RNA
primer
o Lagging strand requires numerous
RNA primers
 RNA Primer is created by DNA primase
o Created RNA primer approximately
11 +/- 1 nucleotides in length
o Complementary base pairing rules hold
 The RNA primer is extended by DNA polymerase
o RNA segment is removed by the enzyme ribonuclease H
o And substituted with dNTPs by DNA polymerase δ (delta)
 Synthesis of each Okazaki fragment ends when DNAP runs into RNA primer attached to
5’
 However, a break in the DNA strand remains between 3’ of new DNA fragment to 5’ of
old DNA fragment
o Sealed up by DNA ligase

Prokaryotic DNA Replication

 Origin of replication (OriC) is ~245 base pairs in length
o Prokaryotes have one distinct OriC while eukaryotes have multiple
o Consists of a DnaA binding region (TTATCCACA) and an A-T rich region
o DnaA binding region has several 9 base pair DnaA binding repeats, and the A-T
rich region contains several 13 base pair A-T rich repeats
 In order for DNA replication to begin, the 2 DNA strands must be separated
o Happens in the A-T rich region
 Since A-T bonds only have 2 hydrogen bonds, and are weaker than G-C bonds, it is
easier to separate
o This region is opened by the binding of the DnaA to the DnaA binding region
o DnaA is a homotetramer, composed of 4 identical monomers
 DnaA proteins bind to the DnaA binding region, causing stress on DNA strands
o Leads to strand separation
o There are also single stranded binding proteins (SSBP) preparing to bind the now
single stranded DNA (ssDNA)

 SSBPs bind to ssDNA to prevent them from re-annealing

o SSBPs are also homotetramers, being composed of 4 identical monomers
 Once the ssDNA has been exposed, the replication machinery can begin to assemble
 DnaB, also known as helicase, arrives first
o Helicase recruits other proteins and is central to the process of replication
 o Helicase is a homohexamer, with the N termini of all six units facing the same
side, opposite to their C termini
o Helicase resembles the shape of the lock washer and can assume an open or
closed conformation
 Can only load onto ssDNA when in open conformation
 When not bound to DNA, helicase is found in complex with DnaC, its inhibitor
 The helicase-DnaC complex has interaction between helicase and DnaC,
o Causes helicase to assume open conformation
 Notice the central channel formed in the open complex, which allows one strand of
ssDNA to pass through it during DNA replication
 DnaG (primase) now arrives
o Primase comprised of 3 domains
o A DNA dependent protein
 Primase binds to N terminal domain of helicase, opposite of the side of the inhibitor,
DnaC
 The two primase-helicase-DnaC complexes are oriented in opposite directions

 DnaA interacts with

one complex through DnaC, and the other through helicase
 The antiparallel structure of DNA explains the opposite loading of the complexes on the
strands
 Helicase and DnaC are still in the open formation at this period

o As DnaC
detaches from the complex, helicase assumes the closed conformation
o Both of the primase-helicase complexes detach from DnaA, while one complex
moves to the opposite fork
  DnaA is now no longer
needed, so it dissociates from the complex
 Now, there are 2 replication forks
o Each one has a helicase and a primase ready to recruit other proteins necessary to
form replisome

Replisome Assembly
 Separation of 2 DNA strands leads to formation of a replication bubble
 Each end of the bubble is referred to as replication fork
o Each fork consists of DnaB (helicase) and DnaG (primase), with SSBs bound to
the ssDNA
 The two helicase on either fork move along the ssDNA towards the forks’ junction
o Makes replication bubble larger
o Further unwinds dsDNA
o More room for replisome formation
 As the helicase unwinds, each primase lays down two sets of RNA primers
o When helicase unwinds, the replication bubble increases in size
 Helicase uses ATP to unwind
 Tension through dsDNA forces it open
o RNA primer nucleotides are added
 10 bases in total
o 2 primers are synthesized on each ssDNA strand, to be used later in the
replication process
o In the lagging strand, there is a small RNA primer with the helicase in a 5’ to 3’
direction and the DNAP going 3’ to 5’
 The primer flips and reorients the polymerase
 Now, both polymerase and helicase go in the same direction
 The next protein to join is the clamp loader
o Responsible for loading the DNAP clamp onto ssDNA
o Composed of 3 proteins:
 Gamma
 Delta
 Delta prime
o Clamp loader is considered a pentamer because gamma subunit is composed of an
additional two subunits, that resemble the delta and delta prime subunits
o Loading of DNAP onto DNA requires a gamma complex and a tyrosine proteins
in order to recruit the 2 halves of the sliding clamp
o Assembles onto the primer with a small extension sticking out
 TauC is a subunit of the clamp loader
o Attached to the clamp loader via flexible linker
o Two TauC connect clamp loader to replisome
 Binds to helicase (clamp loader binds to replisome via TauC)
 The clamp loader connects DNAP3 to DNA
o Clamp loader opens up clamp and loads it onto DNA
 Clamp loader + clamp complex has two different conformations
o Loading of DNAP onto DNA requires a gamma complex and a tyrosine proteins
in order to recruit the 2 halves of the sliding clamp
o In bacteria, the clamp is a homodimer
 Core enzyme structure displaces the clamp loader
o Entire polymerase structure is now stabilized by the clamp
Figure 1: Clamp loader has threads matching grooves in DNA. It binds to a free sliding clamp, The structure of the clamp loader
(dark green) resembles a screw nut, with its threads matching the grooves of double-stranded DNA. The loader binds to a free
clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. The clamp loader, thanks to its
screw-nut structure, recognizes the region of DNA that is double-stranded and latches onto it, tightening around the complex of
a template strand with a freshly synthesized elongating (primer) strand. It carries the clamp along this double-stranded region
until it encounters the 3ʹ end of the primer, at which point the loader hydrolyzes ATP and releases the clamp, allowing it to close
around the DNA and bind to DNA polymerase. In the simplified reaction shown here, the clamp loader dissociates into solution
once the clamp has been assembled. At a true replication fork, the clamp loader remains close to the polymerase so that, on the
lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment

 Problem: what prevents the polymerase from dissociating from clamp without having to
impede its rapid movement along DNA?
o 3 dimensional structure of clamp protein has it form a ring around DNA
o One side of ring binds to back of DNA polymerase
 Whole ring freely slides along DNA as polymerase moves
 DNAP3 binds to TauC as the last protein involved in replisome assembly
o Each clamp loader loads 2 clamps onto ssDNA and helps replisome take shape
 As first clamp moves down the ssDNA, clamp loader prepares second clamp
o Passes second clamp-ssDNA complex to adjacent DNAP3
o Another DNAP3 joins at the location of an RNA primer
 New clamp-DNAP3 complex uses an RNA primer as the starting point for DNA
synthesis
 On lagging strand template, each
time polymerase reaches 5’ end of
 the preceding
Both replisomesOkazaki
are fragment,
the polymerase
now complete and is released; this
molecule
ready thenDNA
to begin associates itself with
new clamp assembled onto RNA
replication
primer
o At ofeach
next Okazaki fragment
replisome,
dsDNA is
separated into
ssDNA by
helicase
 One strand goes through central channel and into adjacent clamp-DNAP3
complex
 One strand enters second clamp-DNAP complex
o Two replisomes head in opposite directions, replicating the DNA until they meet
at circular DNA genome

 In E. coli, replication bubble opens, two replication forks going opposite strands
o E. coli is a covalently closed circle with no free 3’ to 5’ ends to it
o It will move along two directions
 Replication completes as it moves along through the fork and two circular
daughter DNA molecules form
 In eukaryotic chromosomes, they are linear
 o Free 3’ hydroxyl and 5’ phosphate strand
o Problem for eukaryotic organism arises as the DNA primase cannot bind at the
very end of the chromosome
 RNA primer and incomplete newly synthesized lagging strand following
along the template strand complex
 Fail to replicate a chunk of chromosome
o Go through another cycle
 Chopping chromosomes to make it smaller and
smaller, losing genetic information
 This is not good, as cells are unable to cope
without chopping down
 Solution: Telomerase enzyme can bind to the end of a chromosome, alternative type of
polymerase which extends the end of a chromosome outwards
o A different type of DNA polymerase can now bind to the strand
o DNA primer can extend out 5’ primer and extend in the 5’ to 3’ direction
o Enzyme called telomerase has nucleic acid ends in its own internal structure
o A protein that has a small case of nucleic acid
o Telomerase uses the small piece of RNA in itself as a template to extend the 3’ end
of the parental strand
 Results in extending the sequence and its repetitive over and over
 Thousands of repetitive sequences
 RNA is in a position where polymerase can be placed
o Telomerase is an RNA polymerase that requires DNA
 It uses RNA as a template to synthesize DNA

Helicase and Gyrase

 Helicase uses energy from ATP to undergo conformational changes within its six
subunits
o Allow for movement down DNA strand, unwinding dsDNA ahead
 Causes tension on double helix
o When enough tension accumulates, DNA forms a supercoil
 *tangled phone cord*
 o However, gyrase prevents accumulation of tension
 Gyrase is composed of several polypeptides
o Two copies of each polypeptide from form a dimer
 The only components of gyrase structure not solved are the two flexible linkers
 Gyrase complex contains 2 channels
o dsDNA passes through these channels
 ATP – driven conformational changes cuts one strand in order for the other to pass
through
 Helicase unwinding induced tension causes dsDNA to twist around itself
o Each twist results in a crossover  more crossovers  more tension


Gyrase prevents this by cutting open the bottom strand, so the top strand passes through
without creating tension or crossover

 When top strand passes through,

bottom strand covalently joins back
together
o Top strand is eventually
released
 As tension accumulates progressively,
this process continuously cycles until
replication is complete
o Passing strand keeps twisting
and looping up behind gyrase
to attempt further crossovers
 In supercoiling DNA:
o Without gyrase:
 Crossover occurs
 Twisting continues  additional crossover  accumulates tension
 Supercoiled DNA forms, which needs to be uncoiled
  Done by DNA gyrase and topoisomerase, which decoils and
denatures DNA
 Relieves tension by making single and/or double stranded breaks
in supercoiled DNA
o With gyrase:
 Prevents crossover by allowing one strand to pass through the other
 No tension accumulated
Lagging Strand, DNAP1, and Ligase in Replication
 In leading strand, incoming nucleotides are triphosphates are added onto the 3’ end of the
growing chain
 DNAP3 breaks bond between these phosphates to release energy
o Released energy is used to add nucleotide onto growing chain
 Since synthesis proceeds in a 5’ to 3’ direction:
o 5’ phosphate attached to 3’ OH end of previous nucleotide
 Lagging strand runs antiparallel to leading strand
o Lagging strand polymerase doesn’t change orientation
o Instead, lagging strand DNA enters polymerase from opposite end
  Lagging strand enters the central channel of helicase and exits at back of replisome
 While existing, primase adds a 10 RNA base primer
o Necessary for DNAP3 to begin adding nucleotides at lagging strand
o SSB is still bound to DNA at this time
 After, the clamp is loaded onto the lagging strand
o Complex is passed onto adjacent polymerase
 Lagging strand is folded to bring the lagging strand DNAP into a complex with the
leading strand DNAP
o Also brings 3’ of each Okazaki fragment close to start site of new Okazaki
fragment
 This entire cycle repeats every ~1000 bases
o This entire ~1000 base section is an Okazaki fragment
 After polymerizing the Okazaki fragment, DNAP1 removes the RNA primers
o First removes the RNA with its exonuclease domains
o Then adds DNA with its polymerase domain
 Ligase then joins last base added by DNAP1 with the next base upstream
DNA Ligation
 DNA ligation occurs in two steps
o DNA ends must collide and stay together long enough for ligation to occur
 Easier at lower temperatures due to lower molecular velocities and
stabilized hydrogen bonding between nucleotides
o DNA ligase catalyzes the joining of the 3’ OH of new DNA fragment to 5’
phosphate of old DNA fragment
 ATP binds to ligase and becomes adenylated into AMP + PPi
 AMP nucleotide (attached lysine residue on ligase active site) is
transferred to 5’ phosphate
 AMP phosphate bond is attached by 3’ OH (think back to splicing – how
the OH attacked the phosphodiester bonds during transesterifications)
 Forms covalent bond and releases AMP
DNA Mismatch Repair
 When a mismatched nucleotide occurs, the system can recognize the mismatch
o Base pairs won’t line up, creating a distortion in the double helix
 Strand distinction mechanism in E. coli depends on methylation of selected A residues in
DNA
o Methyl groups are added to all A residues in a GATC sequence
 Not until sometime until after A has been incorporated into the newly
synthesized DNA chain
o Thus, the only non – methylated
A in GATC sequences are new
strands behind a replication fork
o Recognition of non – methylated
GATC allows for its removal
 3 step process:
 Recognition of
newly synthesized
strand
 Excision of non –
methylated portion
 Synthesis of
correct segment
Gene Regulation and Expression
 Protein production requires energy
o Proteins are produced only when needed to conserve energy
 Proteins can be produced and regulated at the level of gene transcription
o Gene is only transcribed when its corresponding protein is needed
 Glucose is a six carbon sugar used by E. coli as an energy source
o Preferred source of carbon
o Can exist as either a free molecule or either as part of a larger carbohydrate
polymer
o E.coli can extract and subsequently digest the glucose molecule found in lactose
by first cleaving off the galactose sugar
 Less efficient than starting glucose digestion with single, glucose
monomers
o Lactose only metabolized if glucose is absent
 Lactose is made of a single glucose and single galactose
 Converted into something E. coli can absorb
 Thus given the choice, E.coli would rather digest a single glucose molecule directly than
have to go through the extra step of cleaving it out of lactose
 If glucose molecules are nowhere to be found and lactose is abundant, E.coli will
transcribe the genes and translating the proteins necessary for cleaving glucose out of
lactose
 Main idea: only turn on transcription only when genes are necessary
o As not to waste energy
 Cell must be able to detect presence/absence of glucose and lactose to code for lactose
digesting proteins
 4 combinations
o If glucose is present and lactose is present:
 Cell prefers to digest glucose
 Cell chooses to digest free glucose molecules rather than cleave off lactose
 Protein involved in lactose digestion are thus not required
 Genes not transcribed
o If glucose is present and lactose is not:
 Essentially the same as above
 If glucose is available, then cell will always digest glucose
o If glucose is absent and lactose is present:
 Cell has no choice but to digest lactose
 The only scenario in which lactose digesting proteins are transcribed
o If glucose is absent and lactose is absent:
 Despite lack of glucose, lactose is also absent
 Protein involved in lactose digestion are thus not required
 Genes not transcribed
Lac Operon

 ***A lot of people are confused by the negative and positive control Espie mentioned a
lot in class
o Negative control forms of transcription regulation is anything that inhibits
transcription of lac operon and/or prevents binding of RNA polymerase by
decreasing efficiency of promoter
o Positive control forms of transcription regulation is anything that improve the
efficiency of the promoter by improving the binding of the RNA polymerase
 3 genes are responsible for coding proteins that help with lactoseimport and digestion:
o lacZ
o lacY
o lacA
o These 3 genes exist together as an operon
 Known as the lac operon
 An operon is a cluster of genes under the control of a single promoter
o Transcribed as a single mRNA
 LacZ gene product is β-galactosidase
o Enzyme that hydrolyzes the β-galactoside, galactose, by cleaving the bond
between galactose and glucose, thus freeing the glucose molecule
 LacY gene product is lactose permease
o Inserts into E. coli cell membrane where t creates a channel allowing lactose to
enter the cell
o Energy used to import comes from difference in proton concentration throughout
the cell membrane
o Lactose flows into the cell with the protons
 Allows for passive diffusion(?) of lactose
 LacA gene product is galactoside acetyltransferase
o Function is unclear
o Fucking great, why do we have to know this then?
  Initially, LacY and LacZ genes are not transcribed
o Low amount of LacY in cellular membrane
o When intracellular environment senses the presence of lactose:
 If there is an absence of glucose:
 A change in gene expression occurs
 Upstream of these three genes lies the lac operon promoter
o Site of RNA polymerase recruitment during transcription initiation
o Several regulatory elements lie in the vicinity of the promoter
o Lac operon transcription is regulated by proteins that interact with these
regulatory elements

Lactose Detection

 Mechanisms for lactose detection and regulation of gene transcription are very direct
o Fuck off no they are not, they are confusing as fuck
 In absence of lactose, a repressor protein (known as LacI) binds to specific regulatory
regions of DNA
o These regulatory regions are referred to as operators
o LacI repressor proteins are tetramers composed of 4 identical monomers
 i.e. dimer of dimers (dimeception?)
o Each monomer has its own DNA binding domain, a central dimerization domain,
and a tetramerization domain where 2 dimers can interact
o DNA binding domains of repressor bind to operators at the major groove
 3 operators in the lac operon:
 O1, O2, O3 (O1 and O3 are auxiliary operators)
 One dimer binds to O2, while the other binds to O1 or O3
 This interaction twists DNA into a loop which physically blocks
the transcription by RNAP
 Tetrameric domain where 2 dimers interact binds to specialized sequences
around promoter
  In order for polymerase to bind and transcribe, repression must be relieved
o Homotetramer structure (active) is structured in a way where tails cross over one
another
 Helps hold complex together
 Four zinc fingers protrude to the top, projecting out of the motif and
interact with the major groove
 All monomers are interacting with each other
o A single zinc finger wraps around the major grooves of DNA
 Singular residue points inwards and base pairs with structure
 Stabilizes structure
 Stable structures requires removal of repressor before operon can be
transcribed
 Else, transcription will occur at very low level
 Notice how operator DNA sequences are palindromic
o A result of the repressor amino acids that interact with the first half of the
operator sequence being in reverse orientation as the amino acids that interact
with the latter half

 When lactose is present, lactose binds to repressor

 o Induces 3D conformational change
 Prevents repressor from interacting with DNA
 Repressor then leaves DNA, and lac operon if free to be transcribed
 Release of repressor allows DNA to go to natured state, allows for binding
of RNAP

Glucose Detection & Catabolite Activator Protein (CAP)

 Glucose is detected indirectly

o Via activator of gene transcription
 When glucose levels are low, intracellular cyclic AMP levels are high
o In this case, cyclic AMP binds to and activates CAP
 CAP is an activator of gene transcription
o CAP is a dimer of composed of two identical monomers
 When CAP binds with cyclic AMP, it undergoes a conformational change
o Allows for interaction with a regulatory region close to lac operon promoter
 Referred to as CAP binding site
 o Presence of cyclic AMP bound CAP at the CAP binding site promotes
recruitment of RNAP to the promoter
 Promotes transcription of lac operon
 Although noncyclic AMP bound CAP at the CAP binding site can still initiate
transcription of lac operon, it is at a much lower extent

This change in the lac repressor is caused by the small molecule

allolactose, an isomer (rearranged version) of lactose. When lactose is
available, some molecules will be converted to allolactose inside the
cell. Allolactose binds to the lac repressor and makes it change shape so
it can no longer bind DNA.

Recap of Lac Operon

 Lac Operon is expressed at high levels if:

o Glucose must be unavailable: When glucose is unavailable, cAMP binds to CAP,
making CAP able to bind DNA
 Bound CAP helps RNA polymerase attach to the lac operon promoter
o Lactose must be available: If lactose is available, the lac repressor will be released
from the operator (by binding of allolactose)
 This allows RNA polymerase to move forward on the DNA and transcribe
the operon
 Going back to our 4 combinations:
o Glucose present, lactose absent: No transcription of the lac operon occurs
 Lac repressor remains bound to the operator and prevents transcription by
RNA polymerase
 cAMP levels are low because glucose levels are high, so CAP is inactive
and cannot bind DNA
o Glucose present, lactose present: Low-level transcription of the lac operon occurs
 The lac repressor is released from the operator because the inducer
(allolactose) is present
 cAMP levels, however, are low because glucose is present
 Thus, CAP remains inactive and cannot bind to DNA, so transcription
only occurs at a low levels
o Glucose absent, lactose absent: No transcription of the lac operon occurs
 cAMP levels are high because glucose levels are low, so CAP is active
and will be bound to the DNA
 However, the lac repressor will also be bound to the operator (due to the
absence of allolactose), acting as a roadblock to RNA polymerase and
preventing transcription
o Glucose absent, lactose present: Strong transcription of the lac operon occurs
 The lac repressor is released from the operator because the inducer
(allolactose) is present
 cAMP levels are high because glucose is absent, so CAP is active and
bound to the DNA
 CAP helps RNA polymerase bind to the promoter, permitting high levels
of transcription

The

Nucleus and Eukaryotic Chromosomes

 Nucleus is central organelle

o Stores cellular DNA
 Not aware how it stores it
o DNA doesn’t hang out in nucleus
o It is a very dynamic area
o During cell division, nucleus vanishes
o Highly organized organelle
 o Level of organization is not obviously visible
 Nucleus is surrounded by 2 distinct phospholipid membranes
o Inner nuclear membrane
o Outer nuclear membrane
o Referred to as nuclear envelope
o Outer membrane is continuous with endoplasmic reticulum
 Free space allows for protein communication
 Nucleolus is where ribosome chains are located
o Ribosomal RNA genes are congregated in the nucleolus
o Where rRNA is transcribed
 Regions on inside periphery of nuclear envelope are slightly more electron dense
o 2 distinct layers
 Nuclear lamina
 Intermediate filamentous protein structures that form internal
cytoskeleton
 Gives nucleus shape and offers mechanical support
 Peripheral heterochromatin
 DNA present in the cell that is highly unlikely to be transcribed
 Genes are actively transcribed in nucleus central plasm
o Chromatin is found in nucleus central plasm
 DNA + associated proteins = chromatin
 Nucleus has great big ‘holes’ in its nuclear envelope
o Known as nuclear pores
 Protein complexes forming sophisticated channels allowing for cell
material traffic
 Distinct in nucleus
 Mitotic apparatus is also present
o Help separate chromosomes
 In humans, there are about 3.2*109 nucleotide pairs
Eukaryotic Chromosomes

 Vast amounts of DNA compacted into a dense structure

o If not condensed, equivalent amount of DNA in a chromosome would stretch over
6 feet in length!
 Minimal amounts of protein coding information
 Composed of:
o Highly coiled DNA
 Coiled by specialized proteins that bind to DNA and fold it
 Generated a series of coils and loops providing higher levels of
organization
o Histones
 Highly basic
 Rich in R and L
o Non-histone chromosomal proteins
 i.e. DNAP and RNAP, transcription factors
o Topoisomerases, histone modifying proteins
 3 fundamentally important regions in chromosome structure
o At end, we have telomere/telomeric sequences
 During replication, allows platform for replisome to bind to
 Telomerase enzyme extends chromosome outwards
o Origins of replication
 Multiple in eukaryotes
o Centromeres
 Fuck this class
 Espie is ass
Chromosome Organization

 DNA double helix  Nucleosomes  Chromatin

fiber  Looped domains  Condensed section of
chromatin (heterochromatin)  metaphase
chromosome
 DNA is chromosomes has a total packing ratio of
around 7000
 The first level of packing is achieved by the
winding of DNA around a protein core to produce
a "bead-like" structure called a nucleosome
o Packing level of 6
o Invariant in chromatin of all types
o Makes nucleosome
 The second level of packing is the coiling of beads
in a helical structure called the 30 nm fiber that is
found in both interphase chromatin and mitotic
chromosomes
o Packing level of 42
 The final packaging occurs when the fiber is
organized in loops, scaffolds and domains that give
a final packing ratio 9450 (15*15*6*7)

Histones

 Binds to DNA and is responsible for forming

eukaryotic chromosomes and making my life ass
o Responsible for first level of DNA packing
 Nucleosome
 Can be seen as a series of beads on
a string if partially unwound
o Nucleosome = nucleosome bead + linker
DNA
 Repeat at intervals of 200 nucleotides
 Each core bead consists of 8 histone proteins
 Nucleosome bead
 1.8 turns of DNA wind around protein core
 82 nucleotide pairs per turn (147bp)
 Amino acid sequence is highly conserved
o Organization of DNA is vitally important
 Total histone mass ~~ DNA
 Histones H2A, H2B, H3 and H4 are around 102 to 135 aa each
 o 2 molecules each comprise a nucleosome bead
 Histone H1 is 220 aa, much larger
o Forms higher order structures
 Histone folds (structural motifs of histones formed by three alpha helices connected via
loops) bind onto one another to form dimers and tetramers
o Dimers and tetramers combine to form octamers
 60 million molecules of each histone
 Each histone core contains N terminal tail
o N terminal tail is highly basic (high proportion of R, L, S)
o Modified by addition of methyl groups and acetyl groups and phosphates and
methylenes
o Extends out from DNA histone core
 Globular domain is connected to C terminal tail

Nucleosomes are Dynamic

 Eukaryotic cells contain large variety of ATP dependent chromatin remodeling

complexes
o These complexes include a subunit that hydrolyzes ATP
o Binds to core of protein in nucleosome
 By using the energy of ATP hydrolysis to move this DNA relative to the core, the protein
complex changes the structure of a nucleosome temporarily, making the DNA less tightly
bound to the histone core
o Repeated cycles catalyze nucleosome sliding
 Histone chaperones can remove all or part of a nucleosome core from nucleosome
o Catalyzes exchange of histone dimers
 Thus, histones are replaced on DNA every one or two hours

Chromatin Fiber

 The beads on a string conformation is rarely adopted by DNA

o Rather, nucleosomes pack onto one another to form condensed arrays
o Known as chromatin fiber
o Diameter of 30nm
 The process by which chromatin fibers form is unclear
 Nucleosome-to-nucleosome linkages that involve histone tails, most notably the H4 tail,
constitute one important factor for the tight stacking of nucleosomes
o Furthermore, since histone H1 is larger than normal histones, it is not as well
conserved as other histones, allowing it to change DNA exit path

Looped Domains
  Chromatin fibers can further loop and condense themselves into even higher order
structures
 Genomic regions are flanked by scaffold attachment regions (SAR)
 SAR protein complex form these looped domains
o Each looped domain is around 20,000 to 100,000 bp in length
 Each domain typically corresponds to one gene
 Multiple domains form with one another to associate with a chromosome scaffold
complex
o In turn, this associates with a protein scaffold

States of DNA

 DNA exists in variable packing states along the chromosome

o Heterochromatin
 Most highly condensed form
 Self-propagating
 Represents 10% of interphase chromatin
o Euchromatin
 Variety of extended states
 Often under active transcription
 Enriched in genes
 Each interphase chromosome occupies a particular region
 Organization is thought to be partially due to nuclear lamina
 Nucleolus contains chromatin loops from separate chromosomes

Protein Sorting and Targeting

 Proteins are molecular machines carrying out cell function that all have a destination
 Route from initial translation to final destination is highly regulated
 The largest and most obvious components of cells are the ones that perform protein
trafficking related activities
 Nucleolus: ribosome regulating and producing factory
o Ribosome: protein producing ‘organelles’
 The nucleolus is surrounded by the nucleus
which houses the blueprints for each protein in
the form of DNA
 The nucleus is surrounded by the endomembrane
trafficking system
o Consists of the endoplasmic reticulum
(ER), Golgi apparatus, and transport
vesicles that ferry proteins between
compartments
 Cell structures are highly dynamic and all interconnected
 Incompatible chemical reactions must occur simultaneously in the same cell
o i.e. synthesis and breakdown of proteins
 Segregation is required
o Aggregation into complexes for both prokaryotes
and eukaryotes
 Compartmentalization is only present in eukaryotes
 Proteins must move across membranes
o Single membrane:
 Endoplasmic reticulum
 Golgi apparatus
 Lysosome
 Endosome
 Peroxisome
o Double membrane:
 Nucleus
 Mitochondria
 Chloroplasts
 During protein transport, 2 membranes in the nucleus
must be traversed through
 Proteins destined to be in the cytosol remain there
 There are 3 types of nuclear transport mechanisms:
o Nuclear pores:
 Energy being used to move proteins from cytoplasm to nucleus via nuclear
pores
 Selective entry into nucleoplasm
o Translocators:
 Threading of a folded protein into an
organelle across membrane of organelle
as polypeptide chain snakes through
translocon channel
 Done by protein translocators
 Refolds on other end of protein
 Taken into either chloroplast,
peroxisome, and mitochondria
o Transport vesicles:
 Associated with the endoplasmic
reticulum by a ribosome that is bound at
that location
 Traverses through the ER membrane and enters lumen
 Transport vesicles form and bud off the ER and diffuse down the
cytoskeleton until they reach their destination
  Protein movement has directionality
o ER is a huge warehouse
o Movement has modifications
o Directionality occurs via transport vesicles
o ER transports to Golgi apparatus

Signal Sequences

 Routes by which proteins are sorted and trafficked as well as final destination are coded
o Determined by signal sequences
o Encoded in protein itself at N terminus
o Usually 15 to 60 nucleotides in length that are both necessary and sufficient for
protein targeting
o Cytoplasm is the default final destination
o Once the protein reaches its destination, the signal sequence may be cleaved off
o If it goes to multiple locations, it has different respective sequences that cleave off
o Knowing the sequences gives us clues as to where the protein may be going
 A single polypeptide can have multiple signal sequences

 A factor that determines whether or not a protein will be translated in the endomembrane
trafficking system is the presence or absence of an ER signal
 Proteins with ER signal will be recruited to ER membrane to complete translation
 Those without will be translated in cytosol
  Signal sequences can be relocated if there is an ER signal sequence that allows it to be
targeted by the ER

Nuclear Transport

 Nuclear envelope has an inner and outer membrane

o Continuous but with distinct protein composition
 Inner membrane
o Contains proteins with binding sites for:
 Chromosomes
 Nuclear lamina  protein meshwork
 A protein scaffolding that gives
nuclear envelope shape
 Membrane proteins that help anchor nuclear
lamina
 Outer membrane
o Continuous with endoplasmic reticulum
o Contains the same lipids and proteins as ER
 Nuclear envelope is perforated by many pores that penetrate
both inner and outer membranes
o Each pore is created by a protein complex called the
nuclear pore complex (NPC)
o NPCs create soluble channels that connect cytoplasm
and nucleoplasm
 Makes cytoplasm and nucleoplasm
continuous via aqueous solution
o Ions and other small molecules and proteins can diffuse through the NPCs
o For larger molecules, NPCs act as gatekeepers, regulating entry and exit of
molecules in and out of the nucleus
o NPCs also block the physical diffusion of membrane proteins between the inner
and outer nuclear membrane, allowing them to remain distinct
o NPCs are composed of multiple nucleoporins
 ~ 30 different types of proteins
 Highly similar
 Evolved via duplication (likely)
o We can have these nuclear pores, proteins that maintain architecture within the
pore structures themselves, and anchors that hold it in place
 NPCs are divided into 3 regions
o Cytoplasmic filaments
o Central ring imbedded in nuclear envelope
 Nucleoplasmic ring
 Cytoplasmic ring
  Spoke ring
 Central channel through which cargo travels through is found here
o Protein fibrils point outward into the cytoplasm and nuclear basket that point
inwards into the nucleus
o Small proteins with less than 5.2nm dimeter freely diffuse through NPCs
o Larger proteins must be actively transported via a signal mediated mechanism
o mRNA needs to interact with it and expend energy in order to get in/out

Active Transport

 How does the nuclear pore recognize a protein that needs to be imported/exported?
o Dependent on signals (NLS and NES)
 Nuclear localization signal and Nuclear export signal
 Nuclear pore also has nuclear export receptors and nuclear import receptors
 Precise location in amino acid seems unnecessary
 In a localization signal:
o Machinery doesn’t have to recognize every protein, but it can recognize a small
set of proteins that are
o Destined for the nucleus
o The nuclear import receptor does the recognition of the signal and has a
recognition site for the cargo protein into the nucleus
 In an export signal:
o 4 hydrophobic amino acids that are spaced LxxxLxxLxL
o Spacing is very important
 Cargo proteins that carry larger molecules need a way to transport them through the
membrane
 o Interacts with a shuttle protein that ferries the cargo protein across NPC
 Shuttle is powered by GTP hydrolysis
o Process is known as nuclear transport (how very fucking not creative)
 Ran
o Ran is a small protein
o Used in both import and export
o GTPase (hydrolyses GTP)
o 2 conformations:
 Ran – GTP: active
 Ran – GDP: inactive
 Ran – GAP
o GTPase activating protein
o Triggers the change from Ran – GTP into Ran – GDP
o In order for GTP to be hydrolyzed, RAN needs to been on the cytosolic side of
the membrane and needs to interact with Ran-GAP
o Comes into the nucleus where it encounters RAN-GEF
 Ran – GEF
o Guanine exchange factor
o RAN-GEF converts and extracts the GP and allows the GTP to be brought back
into its binding site
 Pathway regenerates the hydrolysis
 Ran-GDP  Ran-GTP
 Ran-GDP imported to nucleus through its own importin

 Nuclear
localization center has a high binding to the nuclear import receptor, with its cargo
attached to it
o This is the substrate for the nuclear pore
  Ran – GTP
o Promotes cargo unloading for importins and cargo loading for exportins
o Ran-GTP binds to the nuclear import receptor, changing the structure so that the
cargo protein is released
o Get the nuclear import receptor with Ran-GTP attached to it
 RAN-GAP causes the dephosphorylation of GTP to GDP
 When Ran – GTP  Ran – GDP, receptors are freed
 Differential Ran-GDP and Ran-GTP locations confer directionality
 We have our receptor, RNA GDP and the protein that’s destined for export
 In this case, it’s the substrate for the nuclear pore until it gets into a region where it can
encounter Ran Gap which then phosphorylates the GDP
 This re – phosphorylation causes the loss of the cargo and nuclear export receptor
 Every single protein that comes into and out of the nucleus has a mechanism similar to
this

Nuclear Import Steps

1. Cargo protein binds via its NLS to a shuttle

protein “importin”

2. Cargo bound importin binds to a conserved

motif on the cytoplasmic filaments of NPC.
This motif consists of repeating phenylalanine and glycine residues and is referred to as a
FG repeat motif

3. Cytoplasmic filaments hand off cargo

bound importin to central pore channel
4. Cargo bound importin is passed to nuclear
basket
5. Once in nucleoplasm, binding of Ran –
GTP to cargo bound importin triggers
release of cargo protein into the nucleus
6. Ran – GTP importin complex translocate
back to cytoplasmic side of NPC. Once in
cytoplasm, Ran hydrolyzes GTP into GDP
and Pi, causing dissociation from importin.
Importin can now shuttle next cargo
protein
 Nuclear export mechanism is identical but
in opposite direction

Mitochondria is the powerhouse

of the cell
Mitochondria…for real

 Mitochondria synthesize ATP

o Powerhouse of the cell
 Mitochondria has its own genome
o That much of a powerhouse
o Used to be its own goddamn organism!
 That’s like if one of us swallowed another one of us and the person who
got swallowed just became part of the swallower after a couple million
years
o Most of its proteome is encoded by a nuclear genome
 Have a double membrane
o Around its periphery
o Proteins are translocated on both membranes at once
 Mitochondria are sub – compartmentalized
o Matrix
o Intermembrane space
 Continuous with cristae space
 Protein complexes inside create boundaries that create distinction
o These protein complexes are known as TIM/TOM complexes
 Responsible for protein translocation across both membranes
 Membranes in mitochondria are energy inducing
o These membranes can’t have leaks
o The mitochondria doesn’t want uncontrolled current
o Needs that current to generate ATP

Chloroplast

 Dumb and boring

 Plants who cares
 Chloroplasts have ATP and NADPH synthesis, as well as CO2 fixation
 Also has its own genome
o But probably less cool
 Also has a double membrane
o Sounding familiar…
  Sub – compartmentalized
o Stroma
o Ok this is literally the same as mitochondria go look at it yourself

Mitochondrial Translocation

 Multisubunit protein complexes that function as protein translocators mediate protein

movement across mitochondrial membranes
 The first 18 amino acids of the precursor to subunit IV of this enzyme serve as a signal
sequence for import of the subunit into the mitochondrion

 The
signal

sequences that direct precursor proteins into the mitochondrial matrix space are best
understood
o They all form an amphiphilic α helix, in which positively charged residues cluster
on one side of the helix, while uncharged hydrophobic residues cluster on the
opposite side
 The leader sequences of proteins destined for the cytoplasm do not have the same
sequences
 We can make synthetic positively charged amphipathic alpha helices and make them go
into the mitochondria
 Multisubunit protein complexes that function as protein translocators mediate protein
movement across mitochondrial membranes
o The TOM complex transfers proteins across the outer membrane, and two TIM
complexes (TIM23 and TIM22) transfer proteins across the inner membrane
 Along with TIM, is used to move from the cytoplasm into the matrix of the mitochondria
 The TOM complex is waiting for a binding partner
 TIM23 is a translocation process that threads the protein in an unfolded state
o Protein needs to be unbound so that it can go through
o There is a specific enzyme (signal peptidase) that recognizes the leading
sequence, cleaves a particular peptide bond
 It is an endopeptidase enzyme, breaking a specific peptide bond
o The protein folds up into the matrix and carries out its function

 In the above, the protein that’s being made is being prevented from folding
 HSP bind to proteins that have a specific target and prevent them from folding into their
3D structures so that it can be thread through
 The transit peptidase cleaves off the sequence
 The HSP keep it from folding until it’s inside the mitochondria
 Left shows protein translocators in
mitochondrial membranes
 TOM, TIM, SAM, OXA complexes
are multimeric membrane protein
assemblies catalyzing protein
translocation across mitochondrial
membranes
 TOM cannot integrate proteins in
outer membrane
 1st TOM pass unfolded
 Bind chaperones
o Prevents aggregations
 Bind SAM
o Inserts into outer membrane
o Helps with folding
 TOM complex import depends on the hydrolysis of ATP
 TIM22 uses the electrical potential difference to bring it in
 TOM can’t fold on its own, can only act as a targeter in outer membrane
o Due to existence of porins, which are pore forming proteins that aren’t
integratable by TOM
 After translocation through TOM complex:
o The protein is bound to chaperone proteins in the intercellular space between the
inner and outer membrane
 SAM protein complex identifies it, inserts it into outer membrane, and
folds it into its structure
 Outer membrane protein that allows for small solutes to go through it
  Post translocation:
o Pre – entry proteins called precursor proteins remain unfolded
 Due to interactions with other proteins
 Prevented from aggregating
 Signal sequences not always removed

Endoplasmic Reticulum

 Important for most cell’s organelles, including ER itself

 Controls lipid biosynthesis and export
 Controls protein biosynthesis and export
 The plasma membrane of the cell originates as a tissue fragment from the ER
o The small segment of phospholipid bilayer comes from ER
 ER involved in both co and post translational translocation
o Requires signal
 Vary greatly in primary structure, all have a >= 8 nonpolar aa in center
 The ribosomes in cytoplasm are no different than those attached to the endoplasmic
reticulum
 All proteins that go through endomembrane trafficking system go through the ER
o Composed of many interconnected flattened sacs
 Called cisternae
o ER plasma membrane is continuous with nuclear envelope
 Protein synthesis begins in the cytosol and ends in the ribosomes on the ER
 Our ribosomes lacks ER signal and floats or has one that allows it to interact with a
binding partner
 Protein synthesis completed in the cytoplasm and also done in the ER
 Synthesis begins in the cytoplasm; ribosome migrates to ER and protein synthesis is
completed with the sequence being deposited into the lumen of the ER
 There’s another large protein that is the signal recognition particle
o Identifies ribosomes that are in the process of protein synthesis and has a signal
peptide
 Identifies that signal peptide
 The SRP stops protein synthesis in its tracks
 Protein synthesis restarts once the SRP recognizes the translocon on the
ER
 The SRP is a binding partner for the translocon
 Once it’s bound, SRP is released and protein synthesis can
commence
 The protein is being fed through the channel into the ER lumen
 Signal peptidase cleaves of signal peptide
 In the end, we have our proteins that separated from the cytoplasm
 Targeted by its signal sequence and the SRP, which brought it to
the ER surface
 Co – translational translocation begins when N – terminal of protein undergoing
translation contains an ER signal sequence
o Signal is usually hydrophobic, recruits signal recognition particle (SRP)
o SRP is composed of both RNA and protein
 Large complex
 Rod like structure
 Wraps around large ribosomal subunit
 One end of SRP binds ER signal at signal binding site, the other blocks tRNA entrance
channel, pausing translation
 o Also blocks elongation factor site F.12.36
o Gives ribosome time to bind ER membrane
o A safety mechanism to ensure protein is not released into cytosol
o Prevents folding prior to reaching ER
 Entire complex is then recruited by SRP receptor, located in ER membrane
o Complex is handed off by SRP receptor to an unoccupied translocation channel
called translocon
o SRP receptor is a transmembrane protein complex
o Translocator transfers growing polypeptide chain across membrane
 SRP dissociates and translation resumes

 There are 2 classes of

ER proteins!
o Water soluble proteins
 Fully translocated, dissolved into the aqueous phase of the lumen in ER
 Destined for secretion outside the cell
o Enzymes that are destined for other cells
o Trans-membrane proteins
 Either partially translocated during synthesis or integrated wholly into
membrane structure
 ER is main site for synthesis
o A protein gets oriented in the correct configuration
o Reasonably complicated
o Each individual section of a protein also needs to be oriented in the right direction
for it to work
 Can’t change the orientation of a protein once embedded

Transmembrane Proteins
 Proteins destined to be transmembrane in any endomembrane organelles or plasma
membrane remain embedded in the ER membrane
o Inserted in final, correct, functional orientation
o One or more alpha helical membrane spanning segments
 Extra signals required:
o Signal sequence
o Start transfer sequences
o Stop transfer sequences
 Transmembrane proteins pass across membrane
 Initiation requires a hydrophobic start transfer sequence (ala soluble protein)
o Internal “stop transfer” sequence
o A hydrophobic α-helix
o Channel opens sideways
o Signal sequence cleaved
 NH2 terminus + COOH terminus on opposite sides of membrane
o N terminus has catalytic activity at ER lumen, initiates translocation
 additional hydrophobic segment in the polypeptide chain stops the transfer
process before the entire polypeptide chain is translocated
o C terminus has catalytic activity in cytoplasm
o If this particular part of the ER ends up being incorporated into the plasma
membrane, the lumen where the N terminus is becomes the external environment
and the C terminus part stays in the protein
 Oriented such that n terminus points out into environment and c terminus
into the cell
o The ER lumen is “extracellular”, not part of the cytoplasm (can become outer face
of membrane)
o We can position a protein so that it has an integral membrane function
o N and C terminus are soluble
 Anchor catalytic activity to the membrane so that activity occurs on the
surface
 We’ve given this enzyme a location: either inside or outside the cell
 Single pass membrane proteins:
o Membrane proteins spanning plasma membrane just once
o N – terminal ER sequence is initially bound to translocon channel as rest of
polypeptide chain is thread through ER lumen
o When hydrophobic transmembrane domain exits ribosome, it acts as a stop signal
by interacting with residues on inside of channel
 Holds protein in place
 Prevents stop signal from passing through
 Rest of protein forced to stay in cytosol
o When translation finishes, the ER signal sequence is cleaved off by signal
peptidase and transmembrane domain is released horizontally, from the channel
into the ER membrane bilayer
o Newly formed protein diffuses through the ER membrane
 Multi pass membrane proteins:
o Only major difference is that multi pass membrane proteins have additional start
and stop sequences
 Thread through ER like sowing machine
 Can be very complicated (i.e. PSII)
 Note: INTERNAL signal sequences are not cleaved
 If signal sequence is in C terminus:
o The SRP binds to the sequence and it binds to the receptor and threads it through
the translocation channel
 This gets anchored in the translocation channel
 It is thread through until it reaches another stop transfer segment, which
prevents further translocation of polypeptide
o We have a protein that has a number of external loops
Protein Processing in Endoplasmic Reticulum

 Cells can control activity of protein

o Some made proteins don’t initially work and must be activated to work in another
part
 Protein oligomerization occurs in ER
o Multiple covalent modifications of chains
o Proteins destined for secretion are glycosylated
 Adorned with sugars, N linked
 Proteins are sorted and transported or retained
 o Retained in ER – KDEL – COOH
o Vesicular transport to Golgi
 Disulfide bond formation
o Essential for normal structure and activity
o Tertiary and quaternary structures stabilized by disulfide bonds
 Formation is:
o Spontaneous
o Peptide still on ribosome
o Can be sequential (may not)
o Break and reform
o PDI rearranges disulfide bonds to appropriate configuration
 PDI also catalyzes formation of these bonds
o Prevalent in oxidized environment
 Disulfide bond formation stabilizes and activates insulin
o Must be rearranged

 N linked glycosylation
o Large number of sugar residues placed on specific aa residues
 Changes character of protein
o Occurs in ER
o During translocation of protein, in translocation channel, Asn residues in the
chain Asn-X-Ser or Asn-X-Thr triplets identifies N glycosylation location
 Signifies Asn resides will be modified by addition of polysaccharide
 Polysaccharide chain must be assembled in ER
 Requires dolichol
 o Highly soluble in lipid membranes
o End has pyrophosphate residue
 Oligosaccharide transferase is what catalyzes movement of
oligosaccharide from dolichol onto Asn residue
 Now the Asn residue is glycosylated
o Most often, oligosaccharide associated with proteins means protein is destined for
export
 Retained proteins have little glycosylation
 ER retention signal
 Immediate destination of all other proteins is in Golgi
o Transport vesicle moves via diffusion off ER to Golgi
 Fuses with Golgi complex membrane
 Vesicular transport
o Taken from lumen of ER to lumen of Golgi (or lumen of donor compartment to
lumen of target compartment)

 Exit from ER is selective

o Incorrectly folded proteins and incorrectly assembled multimers are retained
 Degraded in ER
 Further attempts
 Vesicular transport
o Provides routes of communication between interior of cell and its surroundings
o Goes outwards
 ER to Golgi to endomembrane system or plasma membrane
o Goes inwards
 Plasma membrane to lysosome
o Highly organized, continual budding and fusing of transport vesicles
 Trafficking through endomembrane system
o See diagram
o Vesicular transport is complex, arduous
  Many formations and disappearance of transport vesicles as process
continues
 Ultimately, protein ends up at secretory vessel
 Anterograde transport
 Diffuses to plasma membrane and fuses, releases contents to
extracellular environment
 Sometimes, movement is in retrograde
 Endocytotic
 Molecules are ingested in endocytic vesicles derived from the
plasma membrane and delivered to early endosomes and then (via
late endosomes) to lysosomes
 Retrieved later on and returned to ER or Golgi
 Surround regions of extracellular enviro
 Creates internal vesicle
 Takes protein cargo to ER
 In short, multiple destinations/just follow the diagram lol

 Needs some clues as to where proteins are coming from and where they’ll end up
o Info resides in vesicle itself
o Budding vesicles have distinct protein coats
 Shapes membrane
 Captures mol for transport
  Only resent for part of life of transport vesicle
 Coat lost after budding
 Exposes membrane
o Clathrin coated vesicles are originated from Golgi, traverses protein to lysosome
o COP coated vesicles are originated from ER, go to Golgi
 Golgi apparatus is also divided into specific sections
 Vesicle budding
o Protein associated with membrane begins to bend and distort shape of membrane
of ER or plasma membrane (wherever it originates from)
o Pinches in to form depression
 Accumulation of cargo
 Either side of membrane begins to fuse together
 Pinched off
 Prevents leak and loss of material
o Vitally important for formation

 For another schematic:

o Cargo assembly proteins are important for cargo selection
o Building the clathrin coated vesicle
o Adaptor molecules bind to face of receptors
 Clathrin is recruited
o Interaction of clathrin distorts membrane
o Vesicle is completely coated
 Dynamin associated region constricts the end of interface between vesicle
and membrane and cleaves it, fusing either end of vesicle
o Away goes everything
 Naked transport vesicle
  Proteins face outwards
 Determines destination of vesicle, where it will bind
 Will find receptor for recognition
 Vesicle migration
o In short distances, it can diffuse
o Over long distances uses active transport
 Through energy use, moves along microtubule towards end of it
 Vesicle docking
o After budding from the ER and transported to its destination, a transport vesicle
must recognize its target and “dock” with it
o Vesicles display markers on surfaces
o Recognized by complementary receptors on targets
o Recognition depends on SNARE
o Fibrous tethering proteins and Rab and GTP hold transport vesicle in place
 FTP tethers to surface of transport vesicle
o RabGTP associates with transport vesicle
o Tethering protein interacts with Rab GTP (Rab effector)
 SNAREs interact and see if match exists
 If not then release and move on
 Else, membrane fusion process begins

 SNAREs
o Vesicle snares (v-SNAREs) are recognized by t-SNAREs (target)
o SNAREs and their structure decide vesicular docking
 Better match creates higher affinity, vice versa
o Mistakes can happen but infrequent
 Vesicle Fusion
o Requires very close approach of 2 membranes
o Energetically unfavorable
o Catalyzed via fusion proteins
o Allows dumpage of cargo into membrane
o Fusion proteins remove water from surface of membrane
 Allows physical and chem components of membrane to be closer
 Water acts as barrier
 Helps membrane of transport vesicle become one with the membrane of
the target compound
 Creates a new phospholipid bilayer with unique protein composition
 Brings integratable membrane protein
 Expands target membrane
o Membranes are very dynamic, constantly changing composition throughout cell
cycle

Golgi Apparatus

 ‘pile’ of flattened
membrane bound sacs
o Set of
membrane
structures
o Capable of forming small membrane vesicles
o Has polarity
 Transport vesicles move content throughout Golgi and is modified as it transports
  Most frequent first destination
 3 regions introduce different modifications
 Many functions are done in the Golgi
o Glycosylation
 Proteins delivered to Golgi are glycosylated by different mechanism
 Uses serine residues to link
sugars to it via O link
o Glycolipids
o Modification of N-linked glycosylation
o Sorting capabilities
 Series of receptors allowing for sorting
of proteins
 Manufacturing of vesicles to target
these proteins

Functions of Glycosylation

 Retained in ER until protein is properly folded, assists

in the folding process and dictates folding process
 Protects from degradation
 Acts as a transport sequence (i.e. lysosome)
o Helps recognize specific residues on sugar
 Cell-cell adhesion in extracellular environment
o Cellular matrix

Golgi sorting and processing

 Cis Golgi
o Phosphorylation of oligosaccharides on
lysosomal proteins
o Removal of Man
 Medial Golgi
o Removal of Man
o Addition of GlcNAc
 Trans Golgi
o Addition of Gal
o Addition of NANA
o Sorting
 Golgi is a fairly stable structure

Inactive Precursors
  Most secretory proteins stored in vesicles as prohormones or proproteins
 i.e. insulin is initially preproinsulin and proinsulin
 Specific enzymatic cleavage generates mature, active molecule
 Preproinsulin has signal peptide cut off
o Sec 11 cleaves signal peptidase at Alanine (signal peptide recognition sequence)
 In insulin, there is catalyzation of disulfide bonds in proinsulin
o Protein disulfide isomerase makes disulfide bonds
 A series of proteases cut the peptide bond which separates C chain from functional
insulin
o Insulin released into bloodstream
o Insulin has two small polypeptide fragments with intra and inter disulfide bonds
 Protease cpe breaks peptide bond at threonine and arginine, protease CP1/3 cleaves at 2
arginine, protease CP2 cleaves at lysine and arginine, happens in secretory vesicle
o Diagram shows locations of cleavage if unclear
 pH in vesicle is 5.5 but pH in blood is around 7
o Release into extracellular environment turns aggregate into more soluble material

 Causes dissolving into soluble protein into bloodstream

 LMAO he took his fucking figures from Wikipedia im schleeep

Membranes

 Membranes are everywhere

 Fuck membranes
 Innovation of eukaryotic cell is proliferation of membranes
o Allowed for compartmentalization
o Massive increase in intracellular surface area
 i.e. allows for ribosome attachment and protein synthesis
 i.e. in red blood cell, we can see 2 track like structures in an EMG
o Hydrophilic regions
 Bind heavy metals
o Origin of the notion that membranes are lipid bilayer
o Outer area is hydrophilic (orients to a water surface)
o Inner area is hydrophobic tails (orients to non-aqueous surface)
 No electrical charge to bind heavy metals
 Membranes have distinct properties
 Most membranes are gel like viscosities
o Not quick solid yet not fluid
 Membranes have distinct composition with proteins that determine to a large extent what
membranes do
o i.e. Sec 11 determines the endoplasmic reticulum structure
 Most common lipids are phospholipids
o i.e. phosphatidylcholine
o Glycerol based
 Starts with core of glycerol
 Alcohols link to phosphate groups
 i.e. choline
o Type of phospholipid determined by:
 Nature of fatty acids at end (nonpolar tails)
 Nature of alcohols covalently linked to phosphate groups
 Heads of molecules can be electrically charged
 Higher temperatures result in more fluidlike consistencies
o Creates leaky membranes (diffusion of molecules, unhindered)
 Phospholipids are not covalently bound to one another
 Multiple hydrophobic interactions are between hydrocarbons
o Strength of interactions decrease with existence of alkenes and alkynes
 Causes kink in hydrophobic tail
 Occupies more volume to create separation of lipid tails
 Interactions increase with increasing length and duration (less fluidity)
 Interactions decrease with decreasing length and duration (more fluidity)
 Lipid composition and structure effects membrane fluidity
o Saturated hydrocarbons create linear structure which creates more hydrophobic
interactions decreasing fluidity
o Unsaturated hydrocarbons create extended structure which creates less
hydrophobic interactions increasing fluidity
o Short trails result in reduced surface area making less hydrophobic interactions
 Increases fluidity
o Long tails result in more surface area making more hydrophobic interactions
 Decreases fluidity
 Thus, something like cholesterol, with double bonds and long tails, decreases fluidity
 Membranes are also asymmetrical
o Phospholipid concentration will vary depending on the location of the membrane
o Movement of lipids in unlikely, very energy demanding
 Membranes are created in the endoplasmic reticulum
o Membrane lipids are inserted into the cytosolic face of the ER
 Transferred to opposite face by an enzyme c
 Process is catalyzed by scramblase in ER membrane and flippase
in plasma membrane
 Can an enzyme called 4.0-ase give me a good fucking mark in this course?
 This establishes an asymmetric lipid distribution
o Lipids are stored in lipid droplets
 Only a single layer of phospholipids
  Membrane proteins define what moves across a membrane
 Membrane components are all interconnected
o Extensive network of proteins that form a surface skeleton
o Integral membrane proteins, peripheral membrane proteins, etc.
 Cell membrane cortex
o Relies in phospholipid bilayer sheath attached to transmembrane protein
structures and small molecules of membrane proteins
o Spectrin links junctions of actin in junction complex
o Cortex structure has membrane microfilament linkages with core actin filaments
 As well as actin filaments and spectrin connecting fibers
 Keratin intermediate fibers hold upper filaments

Cell Surface

 Asymmetrical
 Coated with glycoproteins
 FUCK PROTEINS
 Proteins on external surface of cell are all glycosylated
o Inner surface does not have glycosylated cells
 I don’t care anymore sorry
 Answers for TT3 Take-Home Q’s


 Below is a model of a replisome in the   process of synthesizing DNA at a single

replication fork. The components are labeled A ‐ L. Indicate the name of each
component and state its major function / purpose. [20] 

 

Name Function

A Sliding clamp Protein fold that serves as a promoting factor. Binds DNA
polymerase III and prevents it from dissociating  from the
template strand.

B DNA polymerase III 5’ to 3’ polymerization. Adds DNTP’s to the new daughter

strand by forming phosphodiester bonds.
Has a proofreading mechanism (i.e. 3’ to 5’ exonuclease) 
Powered by triphosphate hydrolysis to PPi
C Single stranded Binds to ssDNA to prevent it from reannealing and stabilize it.  
binding proteins (SSB)

D Leading strand  Acts as a template strand, from which dsDNA is replicated

continuously in the 3’ to 5’ direction through polymerization.

Daughter strand grows continuously (5’ to 3’) by DNAPIII after primase

lays down an RNA primer.

Daughter strand grows by 3’ -> 5’ phosphodiester linkage formation

E Flexible linker  Attaches TauC to the clamp loader.

F DnaB (helicase) Opens and unwinds dsDNA by breaking H bonds. It gives

structural integrity to the replisome and makes its core.
Lock-washer
Loads DNA in open conform. with DnaC, and closes when
DnaC dissociates
Helps recruit other replisome factors

Uses energy from ATP to undergo conformational changes with

in its 6 subunits to allow it to move down the DNA strand

G DnaG (primase)  Adds two pairs of RNA primers which add nucleotides (10bp)
and also break 3 OH bonds  for DNA polymerase III to
synthesize 

H Lagging strand Replicated discontinuously in short sections. Contains okazaki

fragments.

K2 RNA primer Segment laid down by DnaG (Primase) that is extended by

DNAPIII

Gives a platform or DNA polymerase III to bind

 L Clamp loader Responsible for receiving the beta sliding clamp, opening it and
loading the clamp on to ssDNA.

 Describe the structural organization of a nucleosome and the 11 nm chromatin fibre.

[10] 

 In eukaryotes, genome size is far greater than the size of nucleus and fitting the giant genome in
the small nuclear compartment is difficult. Also, successful mitotic and meiotic division of the
nucleus would be impossible without highly-organized condensation of the genetic material to
ensure each offspring receives all of the genes they need for survival. So, to tackle this problem,
the genome is packed into highly ordered chromatin (heterochromatin) by nucleosomes as the
basic repeating unit. Nucleosomes consist of DNA (negatively charged) and histone proteins
(positively charged). Nucleosomes are also dynamic, as they can rapidly unwrap and rewrap to
incorporate for different proteins.

In eukaryotes, the genome size is more large than the actual nucleus size and that fitting the
genome requires a progress. As mitosis and meiosis division would be impossible to overcome
without the the packing and the condensation of the material that is put into the nucleus,
important proteins genetic material would not be able to be processed. Therefore there is
something called chromatin in which is made of many nucleosomes that are packed and
condensed. Nucleosomes contain negatively charged DNA and positively charged histones.
Nucleosomes are also dynamic as they can wrap and unwrap for other proteins.

The first level is the wrapping of the double helix DNA around the histones which form the
nucleosomes. The DNA is 2 nm and the nucleosome is 11 nm with a width of 6 nm. Is has the
appearance of beads on a string due to the clusters that are separate and are linked with short
regions of the DNA strands. The DNA is 40 nm in length and would become 5.7 long after this
process.
 The first level of DNA organization in eukaryotes is the wrapping of the double helix around
histone octamers (this DNA-protein structure is called the nucleosome). The DNA double helix
has a diameter of 2nm, and the nucleosome has an 11nm diameter with a width of 6nm. It has
the appearance of, “beads on a string,” because these nucleosome clusters are separate but
linked by short regions of DNA helices. A DNA double helix of 40mm in length would become
5.7mm long at this level of organization, which gives a 7x packing ratio.

The four different histones of the nucleosomes are H2A H2B and H3 and H4. There are two each
in the nucleosomes which forms an octomer.

The negatively charged DNA then wraps around the octamer 1.8 times and histones are
positively charged due to the lysine and arginine positively charged residues. H3 forms a N
terminal tail which can be used for methylation.
  The 4 canonical core histones that comprise the nucleosome are H2A, H2B, H3, and H4. Each of
these histones are highly conserved across many different species. A nucleosome molecule
contains two of each of these four histone proteins, forming an octamer. 

 The negatively-charged DNA wraps around the octamer core 1.8 times, with 147 base pairs of
DNA per nucleosome (about 82 base pairs per turn). Histones have a central globular domain
with a high proportion of positively-charged residues, like lysine and arginine. The H3 histone N-
termini tails protrude from the nucleosome and are susceptible to post-translational modifications,
like methylation of certain residues for protein interactions.

 These 11nm structures coil into hexagonal hexamers of nucleosomes, which are 30nm in
diameter, and further condense the DNA. H1 histones (not highly-conserved) are bar-like linkers
between the nucleosomes that help stabilize them.

 The nucleosome is the basic unit of organization of a chromosome, which is tightly-wound
chromatin seen at the highest level of DNA organization in eukaryotes during mitosis.





In eukaryotes, the genome size is by far larger than the actual size of the nucleus. Therefore,
there is a process into condensing the genetic material into the nucleus. Mitosis and meiosis
processes would not be possible without the condensing of this material and the important
genetic material would not be processed. Therefore, the genome is tightly packed into something
called chromatin, which is made of many nucleosomes as the basic repeating unit. Nucleosomes
are made of negatively charged DNA and positively charged histones. Nucleosomes are dynamic
in which they can unwrap and wrap for specific proteins.

The first step in this is that the DNA is wrapped around the histones. DNA is abour 2 nm
diameter and the nucleosomes are 11 nm long and 6 nm wide. The DNA about 43 nm long but
after this process it will only become 5.7 long which condensed it 7 times. This shows the
structure as beads on a string looking because of the clusters of nucleosomes and also the short
fragments of DNA between them.

There are four different core histones, H2A, H2B, H3, and ,H4. Each nucleosome has 2 of each
of these core histones forming an octomer.

The negatively charged DNA is the wrapped around the octomer 1.8 times anh these histones are
positively charged as they contain lysine and arginine positively charged residues.