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Lecture 16: DNA Replication
Semiconservative mechanism
- experiment: E. coli cells initially grown in 15N, then transferred into medium of 14N; through
density-gradient centrifugation, pattern suggests semiconservative model
DNA Polymerases Require a Primer to Initiate Replication
- DNA synthesis, like RNA synthesis, proceeds 5' → 3' (formation of phosphoester bond between 3'
oxygen of growing strand & alpha phosphate of dNTP
- primer (preexisting RNA or DNA strand) required to begin chain growth
Duplex DNA is unwound + Daughter strands are formed at DNA Replication fork
- At replication origins ORI: usually A-T rich; replication fork
- helicase unwinds parental DNA strands
- primase, a specialized RNA polymerase, forms a short RNA primer complementary to unwound
template strands
- DNA pol then elongates primer, forming new daughter strand
- topoisomerase I: relieves torsional stress produced by local unwinding of duplex DNA
- leading strand: 5' → 3', can proceed continuously from single RNA primer, in same direction as
movement of replication fork
- lagging strand: cell synthesizes a new primer every few hundred bases; primers elongated in 5' → 3'
direction, forming Okazaki fragments; RNA primer of each Okazaki fragment is removed & replaced
by DNA chain growth from neighboring Okazaki fragment; ligase joins adjacent fragments; primers
are added randomly (not at specific site);
o after NDA synthesis begins at primer, primer is removed by RNAse (this is why RNA is used
as primer, to recognize easily amidst DNA)
o when next Okasaki fragment reaches primer 3' to it, DNA pol adds in nulceotides that were
removed by RNAse
Several proteins participate in DNA Replication
- Replication fork protein A (RPA): ssDNA binding protein that helps stabilize unwound DNA so that
it doesn't form secondary structures that would prevent DNA pol from binding to it; dislodged by Pol
alpha and pol delta
- Proliferating cell nuclear antigen (PCNA): stops synthesis of primer by displacing primase +
polymerase alpha (synthesizes primer); keeps DNA polymerase delta (elongates daughter strand)
complex connected to DNA; falls off when it hits a primer, along with DNA polymerase
DNA Replication occurs Bidirectionally from each origin
- experiment: SV40 DNA's replication bubble shows bidirectional replication; edges of bubble are
symmetrical to ORI and advance in either direction
- two replication forks assemble at a single origin, then move in opposite directions; thus each DNA
strand is leading & lagging
Origins of Replication
- in bacteria: one circular DNA chromosome, one single origin of replication
- in eukaryote: multiple linear chromosomes, multiple ORI = solution to replicate such a long DNA in
a reasonable time
Lecture 17: Chromatin Structure
-
chromosome → chromatin → nucleosome → octamer of histones
histone: most abundant proteins in chromatin\
Structure of Nucleosomes
- DNA component of nucleosomes is much less susceptible to nuclease digestion than is the linker
DNA between them
- nucleosome consists of a protein core w/ DNA wound around its surface
- core is an octamer with 2 copies of histones H2A, H2B, H3, H4 (very conserved structure)
- linker has H1 histones; linker DNA is variable; roughly 200 bp for each one with nucleosome +
linker region
- during cell replication, DNA is assembled into nucleosomes shortly after replication fork passes:
specific chaperones that bind to histones & assemble them together with newly replicated DNA into
nucleosomes
Structure of 30-nm fiber
- most chromatin appear as fibers ~ 30 nm in diameter
Interaction between DNA and histones
- nucleosomes bind all chromatin: not sequence-specific
- phosphate: property unique to DNA, also bp independent; spaced evenly, negatively charged
- thus histones can bind to phosphate (not exclusively): basic amino acids lysine + arginine → histone
is a basic protein; binds acidic DNA, positively charged
Modifications of histone tails control chromatin condensation + function
- flexible N-terminus + C-terminus histone tails: extend from globular histone octameric core; required
for chromatin to condense from bead-on-a-string conformation into 30-nm fiber
- histone tails subject to post-translational modifications: thus they interact between nucleosomes
(protein to protein and protein to DNA) based on lysine
- lysine acetylation: amine at tip of lysine residue modified by acetate to make neutral product that
cannot bind to DNA or negative charges; less-condensed beads-on-a-string conformation conducive
for transcription + replication
- lysine deacetylation: reversible reaction, enzymes deacetylate histones to induce negative charge;
chromatin becomes condensed
Detection of specific DNA fragments by Southern blot (hybridization technique)
- cleavage of DNA with a restriction enzyme
- mixture subject to gel electrophoresis, then restriction fragment present in gel are denatured with
alkaline solution + through capillary action transferred onto a nitrocellulose membrane (blot used
because probes do not readily diffuse into original gel)
- filter incubated under hybridization conditions with a specific radiolabeled DNA probe; DNA
restriction fragment that is complementary to the probe hybridizes, & its location on the filter can be
revealed by autoradiography
Nontranscribed genes are less susceptible to DNase digestion than active genes
- chick embryo erythroblasts actively synthesize globin, whereas cultured undifferentiated MSB cells
do not
- nuclei from each cell type isolated + exposed to increasing concentrations of DNase I
- nuclear DNA then extracted + treated with restriction enzyme BamHI, which cleaved DNA around
globin sequence & normally releases 4.6-kb globin fragment
-
DNase and BamHI-digested DNA subjected to Southern blot analysis with probe of labeled cloned
adult globin DNA, which hybridizes to 4.6-kb BamHI fragment
If globin gene is susceptible to initial DNase digestion, it would be cleaved repeatedly & would not
show this fragment, thus the transcriptionally active DNA does not have 4.6-kb band on Southern
blot
Inactive DNA from MSB cells was resistant to digestion, thus inactive DNA is in a more condensed
form of chromatin that shields globin genes from DNase digestion (unacetylated histone lysine tails)
Nonhistone proteins provide a structural scaffold for long chromatin loops
- in situ hybridization: DNA denatured, then fluorescent probe used to label and incubate a cell in vitro,
then where it binds can be scene under fluorescent microscope
- in situ hybridization: with several different fluorescent-labeled probes (anti-sense oligonucleotide) to
DNA of one chromosome during interphase
o chromatin is arranged into large loops (30-nm chromatin fiber)
o some probe sequences separated by millions of bp in linear DNA appeared reproducibly very
close to one another  anchored to scaffold-associated regions SARs
o conclusion: chromatin organized with some points that are anchored and the rest are much
more flexible (various conformations)
- drawers: loops have drawers that allow retrieval of genes that one needs
- insulators: some scaffold-associated regions functions as insulators, which are DNA sequences of
tens to hundreds of bp that insulate transcription units from each other; proteins regulating
transcription of one gene cannot influence transcription of neighboring gene that is separated by an
insulator
Layers of chromatin packing in chromosomes
- metaphase chromosome: condensed scaffold-associated chromatin 
- interphase: extended scaffold-associated chromatin 
- 30-nm chromatin fiber of packed nucleosomes “beads-on-a-string” form of chromatin  short
region of DNA double helix
Lecture 18: Chromosomes
A Chromosome is heterogeneous (even in interphase)
- Heterochromatin: dark regions, contains more condensed material
- Euchromatin: lighter regions, de-condensed material
Metaphase Chromosome
- condensation of metaphase chromosomes results from several orders of folding of 30-nm chromatin
fibers
- chromosomes that become visible during metaphase are duplicated structures; each metaphase
chromosome consists of 2 sister chromatids, which are linked at centromere (constricted region)
- telomeres: end of chromatid
- centromere involved in mitosis: specific protein complexes build kinetochore, which recruits
microtubules; microtubules separate chromatin in mitosis
- karyotype: the number, size, + shapes of the metaphase chromosomes, distinctive for each species
In Metaphase, Chromosomes distinguished by Banding Patterns + Chromosome Painting
- certain dyes selectively stain some regions of metaphase chromosomes more intensely, producing
characteristic banding patterns, specific for individual chromosomes
-
G bands: produced when metaphase chromosomes are subjected briefly to mild heat or proteolysis,
then stained with Giemsa reagent (permanent DNA dye)
o G-bands correspond to unusually low G+C content; creates a very reproducible pattern
Fluorescence in situ hybridization FISH: method of spectral karyotyping or chromosome painting,
uses probes specific for sites scattered along length of each chromosome
o Probes has different fluorescent dyes; 1 chromosome detected w/ 1 color
o Can detect translocation/breaks via multicolor fluorescence (chromosomes break, then refuse)
Chromosome Painting + DNA Sequencing Reveal Evolution of Chromosomes
- when comparing sequence of human chromosomes with those of monkeys, the genes are 98-99%
conserved  pure DNA is very conserved
- however, the way genes are spread on the chromosome are very different (chromosome breaking and
repairing
- even if genes are completely conserved, crossing them will still not be fertile; explains speciation
Polytene Chromosome: The Drosophila Chromosome
- polytene chromosome starts to replicate many times without separating
- asymmetrical chromosome: centromere is very close to one end; half of the chromosome is
centromere and the rest is other arm of chromosome
- centromere always stays united, while the rest is replicated many times; result is an enlarged
chromosome composed of many parallel copies of itself, greatly increases gene copy number to
supply sufficient mRNA for protein synthesis
- these bands are meaningful because they correspond to condensed chromatin
- localization of specific gene by in situ hybridization
Interphase: heterochromatin + euchromatin
- Heterochromatin: dark regions, contains more condensed material; not active or expressed; regions of
heterochromatin can retract or spray; functions:
o gene silencing (regions where transcription is shut off)
o regions that are not changing: centromere + telomere = always heterochromatic
o protection against mobile movements: keep parts of chromosomes inactive to diminish
exchanges
- Euchromatin: lighter regions, de-condensed material; function:
o active transcription
Elements required for replication + stable inheritance of chromosomes
- origin of replication ORI: at which DNA polymerase + other proteins initiate synthesis of DNA
- centromere: constricted region required for proper segregation of daughter chromosomes
- 2 telomeres: ends; experimentally demonstrated by transfection of yeast leucine
Yeast Leucine Transfection Experiment
- yeast accepts plasmids (circular DNA), which replicates along with yeast’s own division; to ensure
all surviving yeasts have plasmid, yeast cell must be dependant on plasmid for survival +
proliferation
- selection: yeast is mutated so that it no longer produces leucine; plasmid has gene coding for enzyme
needed for leucine production
- autonomously replicating sequence ARS: origins of replication in yeast genome; short sequence ~100
bp, generates progeny + allows inheritance of plasmid; problem is that there is no mitotic
segregration
-
-
centromeric sequence CEN: derived from centromeres of yeast chromosomes, segregate equally to
both mother + daughter cells during mitosis; A-T rich, has specialized histones that recognize
particular DNA sequences, and functions to generate proteins + stick together
telomeric sequences TEL: ligated to ends of plasmids, stabilizes linear plasmid to produce LEU
colonies
o a primer is needed to start replication; primer lands randomly & could land far from end of
chromosome, so sequence between primer & 3’ end of template won’t replicate; solved by
addition of telomere
an assay can be used to identify origins of replication, centromere + telomeric sequences: test random
fragments of DNA inserted in the plasmid
Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes
- telomere: repetitive sequence, in humans and other vertebrates is TTAGGG; sequence can be
recognized using FISH; many thousands of bp long in humans & vertebrates
- DNA pol elongate DNA chains at 3’ end, which requires an RNA or DNA primer; daughter DNA
lagging strand shortens at each cell division
- Telomerase: protein-RNA complex, serves as template for addition of deoxyribonucleotides to the
ends of telomeres; thus telomerase is a reverse transcriptase that carries it own internal RNA template
to direct DNA synthesis
- Telomerase is active only during replication, especially in stem cells, embryo & germ cells; also often
reactivated in cancer cells
Mechanism of Telomerase action
- 3’ end of G-rich strand extends 12-16 nucleotides beyond 5’ end of complementary C-rich strand
- single-stranded 3’ terminus of telomere is extended by telomerase: telomerase contains an RNA
template that base-pairs to 3’ end of lagging-strand template
- reverse transcription: telomerase catalytic site then adds deoxyribonucleotides to position 35 using
RNA molecule as a template
- strands of resulting DNA-RNA duplex then slip relative to each other, leading to displacement of a
single-stranded region of telomeric DNA strand and to uncovering of part of RNA template sequence
- lagging-strand telomeric sequence then extended to position 35 by telomerase again, and DNA-RNA
duplex undergoes translocation + hybridization as before; repeat etc
Lecture 19: Chromatin Modification and Epigenetic Mechanisms
Condensation of Chromatin
- heterochromatin: when DNA is less accessible, most genes are not transcribed; ie centromere,
telomeres, specific regions (regions where chromatin is condensed is often cell-type specific)
- in non-differentiated cells, lots of euchromatin present for flexibility in gene expression
- in differentiated cells, lots of heterochromatin present because cells are highly specialized and do not
require many genes; unneeded genes are “silenced” within heterochromatin
Silencing Mechanism at Yeast Telomeres: telomeres are heteromeric sequences
- multiple copies of RAP1 recognize and bind to simple repeated sequence at each telomere region that
lacks nucleosomes
- SIR3 + SIR4 bind to RAP1, SIR2 binds to SIR4; thus RAP1 recruites SIR2
- SIR2 is a histone deacetylase that deacetylates the tails on histones neighboring the repeated RAP1
binding cite
-
Hypoacetylated histone tails are also binding sites for SIR3 + SIR4, which in turn binds additional
SIR2, deacetylating neighboring histones
Result is chromatin condensation and association of several telomeres; end of telomere is embedded
inside big chromatin structure
Role of Chromatin in gene silencing: mating phenotype in Yeast
- Central mating-type locus MAT determines whether cell has a or alpha phenotype
- silent mating-type genes are located at HML locus; opposite mating-type genes are present at silent
HMR locus; when alpha or a sequences are present at MAT locus, they can be transcribed into
mRNAs whose encoded proteins specify mating-type phenotype of cell
- silencer sequences near HML and HMR bind proteins that are critical for repression of these silent
loci; when mating occurs, one of the 2 genes translocate to MAT
- silencer sequence: not specific for a particular gene or chromosome region; can repress any nearby
gene; if silencer removed, both mating types are expressed simultaneously
- deacetylation is involved in silencing
o mutate histone tail by replacing lysine with arginine (which cannot be acetylated)  silencing
o mutate histone tail by removing positive charges and put glycine instead; this mimics
acetylation  no silencing; HMLα and HMRa were expressed, inhibition of binding to SIR
protein
- RAP1 can also bind silencing sequences: also triggers chain reaction with SIR 2,3,4 proteins; leads to
deacetylation of lysine tails + condensation of chromatin
- In multicellular eukaryotes, condensation is more complicated: methylation + Polycomb complexes
silence whole regions of the genome; these silenced regions can be inherited
Chromatin Immunoprecipitation: identifying chromatin regions containing acetylated histones
- one cannot predict which region of genome is heterochromatin
- an antibody can specifically recognize the epitope that is specific to acetylated or deacetylated
histones; difficulty is that antibody is hard to produce
- isolate and shear chromatin mechanically: breaks down DNA into 2-3 nucleosome frags
- add antibody against a particular acetylated histone tail sequence; bound nucleosomes are
immunoprecipitated (centrifugation, interaction with antibody antigen)
- separate DNA from proteins, then determine DNA sequences bound to the histones (PCR)
Localized regulation of acetylation/deacetylation
- at the level of promoters, similar processes of acetylation + deacetylation occurs
- repression/deacetylation
o TATA box (or any sequence) has specific regions activating or repressing sequences that can
bind to activators and repressors, recruit polymerase, but it can also have effects on histones
o DNA binding domain recruits many proteins; in this case, it is a repressing sequence that
recruits deacetylase which reaches nearby histones b/c of 3D conformation of DNA  URS1,
upstream repressing sequence 1
o Deacetylation also inhibits binding of TFIID
- activation/acetylation
o an activating sequence can recruit reactive component of polymerase & proteins that acetylate
histones, which locally de-condensate chromatin  UAS, upstream activating sequence
o TFIID has evolved to bind acetylated histones (direct effect on polymerase; effect on histones,
which also seem to affect polymerase)
o Forcing acetylation will also produce chain reaction
Modification of Histones
-
-
acetylation: lysine
phosphorylation: serine/threonine
mono-ubiquitination: lysine, add 1 small peptide to end of a tail
methylation/trimethylation: lysine/arginine; irreversible because once you methylate, you cannot
acetylate; to prevent acetylation, heterochromatin protein induces condensation by binding methyl
o enzyme: histone methyl transferase HMT
o 3MeK9-histones recruit heterochromatin protein 1 HP1; HP1 bind to each other  chromatin
compacted; Hp1 recruits HMT  spreading of methylation
more than one modification can occur on the same residue
Chromatin-Remodeling Factors Help Activate or Repress Transcription
- in addition to histone acetylase complexes, multiprotein chromatin-remodeling complexes are also
required for activation at many promotors
- ie yeast SWI/SNF chromatin-remodeling complex: in vitro, the complex pushes DNA into the
nucleosome so that DNA bound to surface of histone octomer transiently dissociates from the surface
and translocates, causing the nucleosomes to “slide” along the DNA
- net result of such chromatin remodeling is to facilitate binding of transcription factors to specific
DNA sequences in chromatin; reversible process, as when proteins dissociate, DNA takes back its
normal configureation around the histones
- many activation domains bind to chromatin-remodeling complexes, and this binding stimulates in
vitro transcription from chromatin templates (DNA bound to nucleosomes)
- chromatin-remodeling complexes also represses transcription by binding to transcription repression
domains of repressors & fold chromatin into condensed structures
Lecture 20: The Genome and General Gene Structure
Genes
- gene: region of DNA controlling a distinct hereditary character; includes entire DNA sequence
necessary for synthesis of functional gene product (ie protein, tRNA, rRNA, miRNA); has promoter,
untranslated regions UTR, and regulatory elements
o mutations can occur at any position in gene region and have very different effects on gene
- bacteria: compact genome, operons; genes produce polycistronic RNA (one promoter, one RNA,
produces several polypeptides)
- eukaryote: genes produce monocistronic RNA (one promoter for each coding sequence; each mRNA
produces a single polypeptide)
o exception: microRNA is polycistronic because the gene is very small
- eukaryotic promoters are regulated at a distance through multiple regulatory sequences
- eukaryotic transcripts are spliced: transcribed RNA (pre-mRNA) contains exons (pieces of coding
sequence) alternating with introns (non-coding sequences) that are removed to produce mature
mRNA; also alternative splicing takes place in 60% of human genes
Human Genome
- size of genome independent of number of genes &/or complexity of organism
- human genome contains much non-coding DNA: transposable elements, repetitive sequences,
unclassified spacer DNA, tandem repeats TR, genes, protein-coding regions of genes (< 2%)
- genes can be split into two groups
o solitary genes: one gene with a particular function; the next gene is remote/far
-
o gene families: group of genes that are closely related, but show divergence and have
specialized functions (ie ECM proteins like collagen, cytoskeletal proteins like keratin in skin,
transcription factors and growth factors)
gene family: gene is duplicated; only one copy is required, the second copy can diverge through a
series of small mutations to acquire new function
Exon Duplication via Unequal Crossing Over leads to creation of new Gene Products
- ie in globin gene, repeat sequence L1 is spread throughout the genome; during meiosis,
recombination between two chromosomes may lead to unequal cross-over
- result of unequal crossing over is that one recombinant chromosome has 2 exons, the other
chromosome has none
Exons are “cassettes” that can be shuffled and used in various numbers and combinations
- example: receptor family of tyrosine kinases
- these transmembrane proteins bind hormones; each receptor has a tyrosine kinase domain, but
receptors at the surface differ (explained by reshuffling)
Tandem Repeats: rRNA, tRNA and snRNA (small nuclear)
- these genes are highly duplicated and have the tendency to repeat
- the duplication is not evolutional; the only function is to help produce RNA
- rRNA produces identical to near identical copies, because the function must be preserved to ensure
the RNA sequence is conserved; proliferating cells need synthesis of millions of ribosomes per day
- rRNA + tRNA required for protein synthesis, snRNAs for mRNA splicing
Single Sequence DNA
- single-sequence DNA: repeats of very short sequences, 3-6% of genome
o satellite (20-100kb), minisatellite (1-5kb), microsatellite (<150b)
- unknown function, no selection; can be varied and have no physiological effect; sequences are highly
conserved in one species, but number of repeats are highly variable from individual to individual
- recombination during meiosis: if short sequences are exactly the same + side by side, likelihood of
crossing over during meiosis is very high; result is different repeat sequences
- minisatellite DNAs are used for DNA fingerprinting through southern blot (hybridization), since each
individual has a different set of these repeats
- also can be used in paternity determination (match patterns in PCR)
Mobile Elements
- mobile elements: ½ of our genome is composed of sequences derived from mobile elements, but most
have been mutated and cannot move anymore
- mobile elements first discovered in corn: gene responsible for corn color can rapidly mutate and
rapidly revert
- some of these DNA/RNA can be packaged in a protein shell (virus), lead the cell and infect other
cells
- transposable elements: DNA transposon and Retrotransposon
- DNA transposon: eukaryotic DNA transposon within flanking DNA move via a DNA intermediate,
which is excised from the donor site; then the DNA intermediate is inserted into target DNA
- Retrotransposon: retrotransposons are first transcribed into an RNA intermediate via RNA
polymerase, then converted back to double stranded DNA by a reverse transcriptase; the DNA
intermediate is integrated into the target site DNA to complete movement
-
-
Various retrotransposons (LTR, LINE, SINE) replicate through distinct mechanisms, but are all
characterized by presence of flanking repeat sequences (anchors) and use of reverse transcriptase
DNA transposon duplication can only occur during replication: transposon can jump ahead of the
fork and replicate
Transposons have no apparent function except self-replication, yet have huge impact
Transposons influence evolution
o Active transposition: brings modifications by interrupting genes or carrying fragments of
genes to new locations; however, most transposons have lost the ability to move by
transposition
o Presence of repeat sequences favors gene duplication, exon duplication and exon reshuffling
by homologous composition
exon shuffling is exchange of exons between 2 different genes; exon duplication is exchange within
the same gene
most of transposons that have landed elsewhere do not move anymore: they lost the ability to be
cleaved (for DNA transposons) or to produce RNA (for retrotransposons); they are dispersed in the
genome at sites where they do not disrupt gene expression
Alu: most common mobile element in human genome, participate in exon shuffling via
recombination between interspersed elements
Extra-nuclear DNA: mitochondria and chloroplasts
- mitochondria and chloroplasts contain DNA coding for some of their proteins, rRNA, and tRNA; the
rest is coded by the nucleus
- mitochondria and chloroplast DNA replicate and multiply by division; they probably originated from
endosymbiotic bacteria
Lecture 21: Molecular Techniques: PCR and DNA Sequencing
Separation of DNA Fragments: gel electrophoresis
- size: easiest way to separate DNA; the only charged group on DNA is phosphate; southern blotting +
hybridization works only with denatured, ssDNA, not dsDNA
- Gel electrophoresis: analyze DNA by size
o Phosphates are negatively charged, thus they move to cathode; small pieces move farther than
big ones
o Ethidium bromide (crystal violet): common dye used to stain DNA; selectively binds to DNA,
making it fluorescent or colored & can be recognized by UV light
o Choosing the appropriate medium: acrylamide or agarose
o Acrylamide gel: used for small DNA fragments (20-2000 bp)
o Agarose gel: used for large DNA fragments (200-20000 bp, roughly the size of a long open
reading frame)
o When concentration of agarose is low, resolution of small fragments is bad; large fragments
are well separated
o When concentration of agarose is high, resolution of small fragments is good; large
fragements are poorly resolved
o If a fragment has a size of 1000 bp, it will migrate as far as another fragment of 1000 bp
because all DNA have ~ the same shapes + properties
DNA Sequencing
- cloned DNA molecules are sequenced rapidly by the dideoxy chain-termination method
-
complete characterization of any cloned DNA fragment requires determination of its nucleotide
sequence
basic idea: synthesize from the DNA fragment to be sequenced a set of daughter strands that are
labeled at one end and differ in length by 1 nucleotide
separation of truncated daughter strands by gel electrophoresis can then establish nucleotide sequence
of original DNA fragment
dideoxyribonucleoside triphosphates ddNTPs lack a 3’ hydroxyl group; thus ddNTPs can be
incorporated into a growing DNA chain by DNA polymerase, but cannot form a phosphodiester bond
with incoming dNTP
add ddNTP to mixture of polymerase action, in addition to normal nucleotides; ddNTP concentration
very low compared to dNTP; synthetic oligodeoxynucleotide used as primer for the polymerization
rxn
give ddNTP fluorescent labels; 4 colors, one for each base
separate by size and count the base pairs; in this reaction DNA has to be denatured by heat or pH
adjustment; polymerization requires a template, primer, DNA polymerase, normal nucleotides +
modified nucleotides at a low concentration
make 4 different reaction mixtures, each with a different ddNTP; put all 4 reactions together on a gel
to get a band of different colors; use detector that reads fluorescence
sequencing of large DNA fragments: use multiple forward/reverse primers, which produces
overlapping sequences
Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture
- PCR: amplifies a sequence by replicating a lot of DNA from a small sample
- Basic process: requires 2 primers to reconstitute double helix: denature DNA by heat  add primers,
nucleotides, and DNA polymerase  complementary strands are produced  re-denature them by
heat  repeat process
- PCR procedure begins by heat-denaturation (95 degrees) of a DNA sample into single strands
- Next, two synthetic oligonucleotides (primers) complementary to the 3’ ends of the target DNA
segment of interest are added in great excess to denatured DNA, temperature lowered to 50-60
degrees
- The primers (high conc.) hybridize with their complementary sequences in DNA sample, while long
strands of the sample DNA remain apart because of their low concentration
- Add temperature-resistant DNA polymerase, which can extend primers at temperatures up to 72
degrees; then PCR procedure is repeated (heat denaturation etc)
- Need to amplify sequence between light-blue and dark-blue primers; sequence is elongated beyond
the limit since only one primer is used at a time; however, with plenty of cycles, the proportion of
elongated sequences will be minimal
PCR Applications
- PCR is a major method to prepare DNA fragment of interest for further applications
- PCR primers can introduce additional sequence: primers don’t need to be identical to the template;
15-20 complementary bp are enough for annealing; thus synthesize an oligonucleotide that is longer;
modification can happen only at the primer
o Introducing a point mutation: if ends of primer anneals and the middle is different, newly
synthesized DNA has point mutation; thus one can verify the nature + location of mutation
via sequencing
o Introducing a restriction site: if half of primer anneals and the other half is different, the
different fragment can be made into a restriction site (recognized by restriction enzymes)
- fingerprinting: combine reverse transcriptase with PCR to detect transcripts
o cell tissue  extract RNA  reverse transcription  mixture of cDNA  PCR using
specific primers  specific PCR product  quantitation/sequencing (ie separate on gel and
compare patterns)
Lecture 22: Molecular Genetic Techniques I: Cloning, Libraries
DNA Cloning
- to prepare large quantities of identical DNA: vector + DNA fragment = recombinant DNA 
replication within host cells  isolation, sequencing, manipulation of purified DNA fragment
- vectors: plasmids are the most common technique in DNA cloning, as it is simple and practical for
small sized DNA; ie bacteria and yeast plasmids
- bacteriophages also used as vectors
- agar plate: soft solid support with nutrients; spread bacteria in a diluted solution where a single
bacteria can replicate into clones (better than tube)
DNA Cloning: Enzymes
- cloning involves cutting with restriction enzymes and pasting with DNA ligases
- principle: open the circular vector and introduce the insert (gene of interest); paste insert into the
vector; vector + insert = new recombinant DNA
- restriction enzyme: cuts at specific sequence; generic name for DNA cutting enzyme is nuclease
DNA Cloning: Restriction Enzymes
- restriction enzymes typically recognize specific 4- to 8-bp sequences, called restriction sites, and
cleave both DNA strands at this site
- restriction sites: commonly short palindromic sequences (restriction-site sequence is the same on
each DNA strand when read in 5’  3’ direction)
- some restriction enzymes make staggered cuts (ie EcoRI, BamHI etc), which produces an sticky
overhang (short sequence symmetrical on both strands); these overhands are complementary +
identical; some restriction enzymes make blunt cuts (ie SmaI)
- Restriction Map: identifies restriction sites of whole sequences; if restriction fragment cut with two
restriction enzymes, it can be ligated in only one possible way
- overhangs hybridize with other overhangs that have been cut by the same enzyme; once hybridized,
ligase recreate the phosphodiester bonds that glue the sticky ends together
- ligating blunt fragments is less efficient: requires energy
DNA Cloning: Engineered E. Coli Plasmid
- plasmids used in molecular biology are highly modified: originally from e. coli, stripped of any
unnecessary sequence; plasmids need origin of replication ORI and resistance (antibiotic resistance
against ampicillin or kanamycin)
- polylinker: multiple cloning site; short sequence crowded with restriction sites for many different
restriction enzymes; with the insert, higher chance of having right enzymes to cut
DNA Cloning: Introducing DNA in cells
- fragment of DNA first purified by PCR; enzymatically insert DNA fragment into plasmid vector to
produce recombinant plasmid
- transformation + selection: insert plasmid into E. Coli in presence of calcium chloride; use bacteria
with a weakened bacterial wall, put resistant gene in plasmid, and make bacteria dependent on
plasmid for growth (culture bacteria on nutrient agar plates containing ampicillin); keep doing
selection at all replication cycles
-
plasmid replication + bacteria replication forms colony of cells, each containing copies of the same
recombinant plasmid
now pick single clone from agar plate and grow it in liquid medium; keep transformed bacteria frozen
for stock, and extract plasmid DNA from bacteria
Preparation of a cDNA Library using Reverse Transcriptase
- cDNA library: collection of complementary DNA, formed from mixture of mRNA
- mRNA characterized by poly A tail (distinguishing factor among all RNA); do reverse transcription
using a primer that recognizes the poly A tail  poly T primer; synthesize DNA using reverse
transcriptase
- crude method, using reverse transcriptase I: digest mRNA with RNases; synthesize complementary
strand of DNA using leftover RNA fragments as primers, which recruits DNA polymerase; 5’ end of
original mRNA is often absent from cDNA library
- refined method, using reverse transcriptase II: digest mRNA, ligate synthetic oligonucleotide (piece
of synthetic DNA single strand primer); now synthesize complementary strand of DNA using the
complementary primer; now the second strand synthesis will always start from the tail of the full
length DNA
cDNA Library
- cDNA library: reverse transcribe as many mRNAs as possible, insert thousands of different pieces of
cDNA in a single vector; clone in plasmids and transform in bacteria
Genomic Library
- similar to cDNA library; contain transcripts (mixture of chromosome fragments)
- difference: chromosome fragments are large and can contain whole genes or parts of genes (exons,
introns, promoters, etc); cloned in bacteriophages
- cDNA library is a complete or partially complete collection of coding sequences, exclusively; no
transcripts
Library Screening
- to retrieve a particular sequence in millions of bacteria, use hybridization
- place bacteria on agar plates, pick clones that are of interest; make a replica by placing nitrocellulose
filter on top to get bacteria on nitrocellulose
- incubate filter in alkaline solution to denature DNA
- hybridize DNA with labeled probe; wash away labeled DNA that does not hybridize to DNA bound
to filter; perform autoradiography
- go back to original plate to pick out the gene
Expression of Recombinant Proteins
- recombinant proteins can be expressed using bacteria
- start with cDNA (coding sequence), which is taken from mRNA
- a promoter is needed to start transcription of the sequence
- note: gene of resistance is also a coding sequence for enzyme that gets rid of antibiotic, thus also
needs a promoter
- introduce plasmid into the cell so that it will be imported to the nucleus and transcribe; no need for
splicing of cDNA sequence, but promoter needed; use viral promoter
- transfection: the act of introducing DNA into cells
- two ways for cells to express protein: transient transfection + stable transfection
Transient Transfection
- place recombinant vector containing cDNA and promoter and viral origin of replicationinto cultured
cells by lipid treatment + electroporation (poke transient hole in cell membrane)
- protein is expressed from cDNA in plasmid DNA; this process is transient because once the cells
divide, some cells inherit the plasmid while others lose it because plasmids are not segregated;
eventually, expression is lost from most cells
Stable Transfection
- in stable transfection, the piece of DNA of interest is permanently integrated into the genome of the
cell
- transformed cell: cell that has been transfected; it is not genetically the same as before
- in most eukaryotic cells rarely integrate new DNA into the genome; since only a few cells that are
transfected with a plasmid will take up the plasmid and fuse it in chromosome, select these cells
- neomycin: antibiotic that kills eukaryotic cells; introduce a gene in the plasmid that will fight this
antibiotic (normally an enzyme that degrades part of it); this way, only cells with plasmid (resistance
to antibiotic) will survive
- G418 = neomycin analogue
- after several weeks, only the cells that have shown continuous resistance are kept; now a stable cell
line is obtained, which will forever express the protein coded for by the inserted gene
Detection of epitope-tagged recombinant protein
- epitope: very precise peptide sequence (8 amino acids) recognized by an antibody
- epitope tag: common way to modify a gene is to add epitope tag to another peptide sequence; this
protein will then be recognized by a specific antibody, and its location in the cell can be determined
Lecture 23: Molecular Genetics Techniques II: Gene Targeting
Gene Targeting
- transgene: stable incorporation of a gene (result of a stable transfection)
o transgenic mouse: mouse that has received a gene that was not there before and is now
incorporated in its genome
- knockout: disruption of a specific gene; impedes gene activity, so gene product is absent
- knock in: replacement of a specific gene; to take out or inactivate one gene and replace it with
another gene
- dominant negative: incorporation of the dominant mutant allele causing the same phenotype as lossof-function; one allele dominates over wild type, inhibits normal gene function
- RNA interference, knock down: depletion of mRNA; gene is still there, but the final amount of gene
product has been decreased; action takes place at level of RNA, not genes
Gene Knockout in Yeast
- yeast has high efficiency of homologous recombination
- to target (remove) a yeast gene, look at the sequence and determine two flanking regions (sites of
recombination)
- PCR: designs a suitable construct for disrupting a target gene: use a selectable marker (resistant gene
like kanMX), use two primers that correlate to both the flanking regions of yeast gene and of kanMX
gene to synthesize disruption construct
-
Transform diploid yeast cells with gene disruption construct, so homologous recombination between
ends of construct and corresponding chromosomal sequences integrates kanMX gene into
chromosome, replacing target gene sequence
Select for G-418 resistance by growing recombinant diploid cells in G-418 medium
If the target gene is essential for viability, half the haploid spores that form after sporulation of
recombinant diploid cells will be nonviable
This technique reveals weather the gene is essential, but not the gene’s function; conditional knock
out studies function
Conditional Knock Out
- if a particular gene is involved in a specific process, then produce lots of yeast and make them
survive up to a certain stage; then implement certain conditions to test the gene’s functions at that
particular moment
- first, delete the gene and transform cell with vector containing gene A under control of GAL 1
(promoter that acts as a switch)
- the promoter GAL 1 is activated in a medium with galactose, which expresses the gene; in a medium
with glucose, the gene is not expressed
Gene Knock In
- to examine the difference and similarity between two genes, take one gene out and replace it with
another one, then examine whether one can functionally replace the other
- one can simply take gene A and modify it by PCR to understand its function
- recombinant protein: there is a possibility that adding a new polypeptide to the end of the protein
might affect its function
- homologous recombination: select homologous recombination in such as way that one does not touch
anything at the promoter, only replace the coding sequence; thus same chromosome location, same
promoter and regulation, but different coding sequence
Gene Knock Out in Mice
- problems compared to yeast cells: haploid stage in mice is very short; homologous recombination in
higher vertebrates is highly infrequent, except during meiosis; in yeast, selection is very simple, but
the same manner of selection cannot be performed in mice; thus knock out must be performed at
diploid stage and produce heterozygous cells
- principle: work with embryonic stem (ES) cells; tricky culture because cells must remain pluripotent;
introduce DNA construct in ES cells to disrupt allele, then inject stem cells in early mouse embryos;
mate mice to obtain heterozygous mice, then homozygous embryos/mice
- formation of ES cells carrying a knockout mutation
o when exogenous DNA is introduced into ES cells, random insertion via nonhomologous
recombination occurs much more frequently than gene-targeted insertion via homologous
recombination
o recombinant cells in which one allele of gene X is disrupted can be obtained by using a
recombinant vector that carries gene X disrupted with neor, which confers resistance to G-418,
and outside the region of homology, tkHSV, the thymidine kinase gene from herpes simplex
virus
o thus in gene-targeted insertion, cells are resistant to G-418 and ganciclovir; in random
insertion, cells are resistant to G-418 but sensitive to ganciclovir
- positive and negative selection of recombinant ES cells
o first, recombinant cells are selected by treatment with G-418 to rid of nonrecombinant cells
o then, the surviving recombinant cells are treated with ganciclovir; only cells with a targeted
disruption in gene X (lacks tkHSV gene), will survive
-
injection of ES cells in early embryos
o inject ES cells into an embryo at blastocyte stage
o embryo has 2 genetic backgrounds: cell from the mother (black mouse, fur color is marker for
genetic background) and ES cells from a brown mouse; they are identical except for the
modifications and the fur color gene difference
o put embryo in pregnant female
o 2 possible progeny: chimeric (progeny containing ES-derived cells) and black
o Chimeric mice (possible germ cells A/X+, A/X-, a/X+) then crossed with black mice (all
germ cells a/X+); ES cell-derived progeny will be brown (A/a, X+/X+ or A/a, X-/X+)
o Screen brown progeny DNA to identify X-/X+ heterozygotes
o Mate X-/X+ heterozygotes  screen progeny DNA to identify X-/X- homozygotes 
knockout mouse
Conditional Knock Out in Mice
- like in yeast, completely knocking out a gene in mice, especially one that is essential, is not very
informative; solution = conditional knock out
- conditional knock out is based on cre recombinase enzyme (from bacteriophage), which can find
specific sequences in DNA called loxP sites
o when cre recombinase enzyme finds two loxP sites, it brings them together and cleaves
whatever is in the middle
- introduce LoxP sites into mice genome in particular locations, creating sites that are ready to be
cleaved off
- cre recombinase gene can be controlled by a promoter so that expression of recombinase will only
occur at controlled stage and place
- loxP-Cre Mouse:
o in one mouse, insert loxP sites flanking the target gene; this is the loxP mouse
o in cre mouse, it is heterozygous for gene X knockout; all cells carry cre recombinase gene,
with cell-type-specific promoter
o cross loxP mouse with cre mouse; part of progeny will inherit the loxP sites and the target
gene, as well as the cre recombinase gene with the promoter
o nothing will happen until cre recombinase is expressed, at which point the target gene will be
excised by recombinase; target gene is thus knocked out
- but this happens only on one allele; difficult to have both alleles knocked out at the same time
o solution: first produce a knockout, then a heterozygous knock out for loxP mouse; thus one
allele is knocked out, second allele has target gene flanked by loxP sites
o as long as the cre recombinase is not expressed, the allele with the target gene intact will
compensate for the absence of the gene on the other allele
Transgenic Mice
- much faster way to modify a mouse is to make a transgenic mouse: simply make it express an
endogenous gene that is normally not expressed (not a gene knockout)
- inject DNA directly into the pro-nucleus, a stage (fertilized mouse egg prior to fusion of male and
femaile pronuclei) that has high chance of random insertion by non-homologous recombination
- transfer injected eggs into foster mother  about 10-30% of offspring will contain foreign DNA in
chromosomes of all their tissues and germ line  breed mice expressing foreign DNA to propagate
DNA in germ line\
Gene Functional Inactivation by Dominant Negative Allele
- in some mutants, only one allele mimics loss of function; however, it seems to inhibit function of the
other allele; this mutation expresses a new gene that is acting as an inhibitor or competitor of the
normal gene
-
-
in many cases, the function of members in a gene family are redundant; knockout of a single gene
will have no phenotype; 2 solutions: multiple knockouts (difficult and laborious), dominant negative
allele
dominant negative allele: produces a mutant form of the protein that inhibits endogenous function;
has inhibitory biochemical function that inhibits many/all gene of a family
example 1: GTPases (cycle between GTP-bound form and GDP-bound form)
o the inactive form becomes active by replacing GDP with a GTP; for this to occur, GEF is
required
o some mutant forms of GTPases act as dominant negatives: they lock into a GTP form by
binding to the exchange factor and preventing all other GTPases from being activated
o this mutant GTPase is a dominant negative construct because by having this gene, it is similar
to knocking out the GEF protein gene
example 2: Hormone Receptors
o found in surface of cells, bind ligands (hormones); many work as dimers and have a kinase in
the cytoplasmic tail, which phosphorylates a neighboring receptor
o as long as there is no signal, those proteins float around the membrane without any interaction
until a hormone binds
o hormone links the two receptors together to form one dimeric receptor  the two receptors
are now close enough for the kinase of one receptor to phosphorylate the kinase of the other
receptor; this phosphorylation triggers a signal
o one can express a truncated form of the receptor such that the kinase doesn’t work; it will
compete with the wild type receptor for binding to the ligand and thus to a second receptor to
form a dimmer; if dimmer is formed with this mutant form, no kinase to activate the other,
and no signal
o when mutant receptor binds to a second receptor, it locks the dimmer in an inactive form,
preventing signals from being triggered
o in a mouse with this mutant dimmer, when the hormone binds and brings together one
receptor with the mutated receptor and the second normal receptor, there is no signal because
the kinase cannot phosphorylate
Gene Knock Down by RNA Interference
- dsRNA is chopped by dicers into fragments, which are used by a machinery to identify and estroy
similar RNA
- 2 methods of introducing dsRNA: in vitro production of dsRNA, RNA interference in culture cells
- in vitro production of dsRNA
o synthesize two plasmids, one containing the sense transcript, the other containing antisense
transcript
o genes are transcribed into RNA and the two strands anneal in vitro  dsRNA
o dsRNA then fed into C. elegans (round worm) and penetrates all cells, including embryonic
cells
o result: target gene is gone; can be seen by in situ hybridization
- RNA interference in culture cells: design of plasmid coding for short dsRNA
o Downstream of a promoter and within the poly linker of the plasmid, prepare a sequence
o Sequence’s first part corresponds to the target RNA, followed by a little “linker” (8 bases),
followed by the inverted complementary sequence of the fist part
o Transfect cells with this plasmid, thus cell produces the RNA
o Due to its sequence, the RNA forms a hairpin structure: the 2 complementary sequences
hybridize and the linker forms a loop
o Dicer cuts the linker of the hairpin strucute and freeze it into dsRNA, leading to RNA
interference
Lecture 24: Protein Structure: Hierarchical Structure
-
protein structure: primary (sequence)  secondary (local folding)  tertiary (long-range folding) 
quaternary (multimeric organization)  supramolecular (large-scale assemblies)
protein function depends on amino acid sequence: aa sequence contains all the info to give 3D
structure and function of protein; primary sequence is direction (has N-terminus and C-terminus); R
groups are the differences between amino acids
Peptides, Polypeptides and Proteins
- peptides = up to 20-30 aa
- polypeptides > peptides
- proteins = natural polypeptide or a complex of polypeptides with a well-defined structure
o reminder: one polypeptide = one RNA translation
Secondary Structure
- random coil: no intramolecular noncovalent interaction between amino acids
- stabilized structures: polypeptides are stabilized by noncovalent interactions, usually hydrogen bonds
- alpha helix and beta sheet: together make up to 60% of polypeptides; also U-turns
- alpha helix: spiral, has a backbone (peptide bonds of polypeptide);
o central spiral is made only of peptide bonds and is held by hydrogen bonds that cement the
spiral in stable structure; H bonds are between carboxyl and amine functions
o Periodicity of 3.6 residues per helical turn; straight rod
o R residues all stick outside, making a sheet around the backbone; interactions with
environment are exclusively dependent on composition of side chains
- alpha helix: in transmembrane proteins, the part of protein crossing the lipid bilayer is always an
alpha helix; the R residues are hydrophobic
- if alpha helix is at the surface of a protein, one face will often display hydrophobic residues that will
interact with other parts of the protein, while hydrophilic residues will be exposed at the surface
(interacting with water and maintaining protein in solution)
o typical periodicity: i, i + 3, i + 4, i + 7 that are hydrophobic
- beta sheet: second most abundant structure, good at making surfaces
o consists of laterally packed beta strands (5-8 residue)
o stability of structure is due to hydrogen bonds within backbone of polypeptide
o residues are either above or below the plain
o residues can be organized so that one property is on one side and another property on the
other side
o conformation at the surface of a protein has a periodicity of 2; hydrophobic residues at
positions i, i + 2, i + 4, i + 8, & polar residues at positions i, i + 3, i + 5
Tertiary Structure
- 4 representations of tertiary structure
o Cα backbone trace: looks only at backbone, demonstrates how the polypeptide is tightly
packed into a small volume
o Ball and Stick: reveals location of all atoms
o Ribbons: more analytical, distinguishes the different structures (helixes, sheets, turns, loops)
o Solvent-accessible surface: map of chemical properties of surface, ie where we find
hydrophilic molecules (+,–,0 charge)
-
different types of proteins: long fibrous proteins (ECM proteins), globular proteins (hemoglobin, βglobin), transmembrane proteins (2 independent sides linked by a helix)
Tertiary Structure: Motifs
- structural motifs: particular combinations of secondary or tertiary structures; contribute to global
structure of the entire protein, and often performs a common function in different proteins (ie binding
to a particular small molecule or ion)
- helix-loop-helix motif/calcium-binding motif: so effective that more than 100 calcium-binding
proteins have the same motif
- zinc-finger motif: found in DNA and RNA-binding proteins; Zn holds alpha helix and beta strands
together; zinc-finger provides negative charges on the sides to bind zinc
- coiled coil motif: motif often involved in self association; present in fiber proteins; two alpha helices
that bind tightly together and make a second-degree spiral; lots of the residues on this motif are
hydrophobic (hydrophobic bonds are short, holding the structure very tightly = dense)
Tertiary Structure: Domains
- structural domain: 100-150 aa-long region, compactly folded, can be made of various motifs; large
proteins are built from several domains
- functional domain: region of a protein that exhibits a particular activity characteristic of the protein;
ie catalytic domain of an enzyme, or regulatory domains, DNA binding domains, and EGF domain
- structural domain: region that often can fold into its characteristic structure independently of the rest
of the protein; ie hemagglutinin HA has two domains  fibrous and globular
- proline-rich domain: because of its aa composition, it functions to bind other proteins
- SH3 domain: motif of several amino acids, found in many proteins; has sequence conservation
Quaternary Structure: Multimeric Proteins
- multimeric proteins can contain any number of identical or different polypeptides
- quaternary structure: the number and relative positions of the subunits in multimeric domains
- ie: hemagglutinin HA is a homotrimer made of three identical polypeptides associated together
- dimmer, trimer, tetramer; homomultimer (subunits are identical), heteromultimer (subunits are
different)
- macromolecular assemblies: association at even higher levels; up to a megadalton in mass; contains
tens to hundreds of polypeptide chains, sometimes also other biopolymers like nucleic acids (ie
ribosomes have inside proteins made of polypeptides, all assembled)
- examples of macromolecular assemblies: transcription initiation machinery, replisome, spliceosome,
nuclear pore complex, components of replication fork (topoisomerase, polymerase, primase, helicase),
proton pump
Evolution of the Globin Gene Family
- Evolution of proteins conserves structure and function, but not primary sequence
- One can change amino acids without changing the structure; ie change leucine for isoleucine (both
are hydrophobic)
- If a point mutation is made in a place that breaks alpha helix or beta sheets, the probability of having
a functional protein is very low because whole structure will break
- Example: hemoglobin (2 alpha, 2 beta), myoglobin, and leghemoglobin (roots of plants, binds
oxygen); primary sequence is very different, but structure + functions are similar
Lecture 25: Protein Structure: Sequence Analysis and Structure Prediction
Sequence Comparison
- read and interpret sequences: some amino acids are perfectly conserved; others have made
conservative amino acid exchanges
- conservative amino acid exchanges: function of residue is maintained
o arginine-lysine: both are positively charged amino acids; differ in shape
o leucine-isoleucine: slightly different in shape; both are hydrophobic residues and of the same
length
o aspartate-glutament: both negatively charged and acidic
- not-so-obvious exchanges
o histidine-aspartate: one is positive, the other negative; if charge is negligent, then one can
replace the other
o serine-threonine: both have –OH in their structure; function of –OH is phosphorylation, so in
phosphorylation enzymes (kinase) there is generally no diff
- hydrophilic aa: basic amino acids lysine and arginine, and acidic amino acids aspartate and glutamate
have strong personalities, since there not few positively and negatively charged amino acids
- proline: hydrophobic residue, makes rigid kink in the backbone of structure
- phenylalanine + leucine have different structures, but both are hydrophobic and thus are considered
conservative exchanges
Structure of a Transmembrane Protein: Ephrin (receptor for hormones or other ligands)
- ephrin has intracellular, transmembrane, and extracellular components
o intracellular: kinase domain, has an enzymatic activity that triggers a signaling cascade;
phosphorylates residues; regulation by phosphorylation (receptors bind together and one
phosphorylates another, which causes cascade)
o transmembrane domain: found by computer, the transmembrane domain makes an alpha helix
in the lipid bilayer (it has the right length and is hydrophobic)
o to determine phosphorylation sites, create a computer program that looks for residues that
could potentially be phosphorylated by a kinase
- the protein folds and makes a scaffold: the surface is what interacts with other molecules; this surface
is dictated by the primary sequence
- mutations in terms of evolution: genetic diseases caused by replacement of one amino acid by another
one with a different property; mutations are selected by evolution and cancer
Lecture 25B: Protein Folding, Modification and Degradation
Protein Folding
- a protein is usually divided into domains, which are basic folding units
- an unfolded polypeptide has polar and nonpolar side chains; the chain folds to hide the hydrophobic
residues from the surface of the molecule and expose residues
Protein Folding: in vitro conversion between native and denatured conformations
- In vitro, place purified protein in urea, denaturing the protein; urea keeps protein unfolded in
suspension
- Remove urea slowly, dilute by dialysis; proteins fold up in correct way and goes back to native
conformation; if unsuccessful, then proteins become misfolded
- Misfolded proteins aggregate
Chaperones/chaperonins help protein folding
- chaperone: binds to exposed hydrophobic residues of nascent polypeptide, helps folding, & protects
from aggregation until properly folded
- example: Hsp70-ATP (Hsp = heat shock protein, accumulate during heat shocks) binds to exposed
hydrophobic residues to maintain polypeptide in motion; Hsp70 protects everything that cannot be in
an aqueous environment
- chaperones are directed by ATP cycle: ATP hydrolyzed into ADP, then replaced by new ATP; makes
chaperones bind and unbind polypeptide
- once chaperone is released from polypeptide, polypeptide can fold properly; if misfolded, chaperones
bind again
- back-up system for misfolded or denatured proteins: Hsp60
- Hsp60: barrel-like structure; recovers proteins that are misfolded before they aggregate together;
recognizes hydrophobic residues that shouldn’t be exposed; these misfolded polypeptides
(hydrophobic residues on the outside) are inserted into the cavity of Hsp60
- Once inside Hsp60 barrel, polypeptide unfolds again; with the help of ATP and GroES, the protein
can fold properly; in this case, ATP is used to regulate the function of the protein itself
Post-Transcriptional Modifications: Covalent Modifications
- acetylation of amine of N-terminus: the free NH2 at the end of the polypeptide is protected from
attack by exopeotidases
- proteolytic enzymes: exopeptidases + endopeptidases
- exopeptidase: cuts at end of polypeptide; ie N-terminus exopeptidase cuts N-terminus of polypeptide
- endopeptidase: cuts middle of polypeptide; these are enzymes important in many mechanisms, ie
lysosomes of cells (degrade proteins) + ECM (for some molecules to cross membrane, like cancer
cells)
- dipeptidase cuts 2 amino acids, tripeptidase cuts 3, polypeptidase cut peptides + oligopeptides
- presinilin: enzyme that cuts proteins in neurons; if not working properly  Alzheimer
- some proteins, like insulin, must be cleaved to function normally
Post-Translational Modifications: Covalent Modifications
- acetylation: acetyl lysine on histones
- phosphorylation: serine, thereonine, tyrosine
- hydroxylation (collagen), methylation, carboxylation
- glycosylation: add sugar to proteins; occurs massively at cell surface or in ECM
o ex. In the cartilage, long polymers of sugars surround protein core; the huge molecule thus
acts like a sponge
Ubiquitination and Degradation
- denatured or misfolded proteins must be rid of
- Ubiquitin: short polypeptide that’s present in all cells in high abundance; attaches to and flags
misfolded protein; Ubiquitin flag is recognized by a proteasome
- Proteasome: degradation machinery; barrel-like structure that denatures the polypeptide into different
small peptides
- This process is used to degrade proteins that are targeted for particular reasons
Neurodegenerative Diseases Caused by Accumulation of Misfolded Proteins
- misfolding: Alzheimer’s, Parkinson’s, and Mad Cow disease
-
principle: a precursor is cleaves and makes a mature protein that is properly folded, but also has a
tendency to switch from alpha helix to beta sheet conformation; these beta sheet proteins aggregate
into filaments resistant to proteolysis
in Mad Cow disease, the change in conformation is infectious: when one protein switches to beta
sheet, it induces surrounding cells to change conformation as well; extremely protease resistant
Lecture 26: Protein Function
Specific Binding of Ligands Underlies the Functions of Most Proteins
- ligand-binding has 2 principles: selectivity and affinity
- selectivity: spectrum of possible interactions for a given protein; determines whether a protein can
recognize only a particular molecule or sequence or whether it can recognize a spectrum of sequences
- affinity: how strongly the molecules bind together; Kd is affinity constant
- ie histones has high affinity for binding DNA, but no selectivity in terms of sequence
Molecular Complementarity: Antibodies
- complementarity determining region CDR: on tip of antibody, the hypervariable region that
recognizes the antigen
- epitope: region of antigen recognized by one specific antibody
- antigen: induces immune response; a single antigen can have many epitopes
- interaction between antibody and antigen may involve many loops and turns on different subunits of
proteins
Enzymes are Efficient and Specific Catalysts
- in a biochemical reaction, one needs to add energy to surpass the threshold of the transition state and
generate product, even if reaction total is favorable
- enzymes accomplish this by lowering energy required for the transition state
- the simplest way to expedite a reaction is to add heat; however, cells are limited to ~37 degrees, as
excessive heat denatures proteins
- example: Protein Kinase A
o enzyme that catalyzes reaction that removes third phosphate of ATP and links it to
polypeptide; ie Serine or Threonine in polypeptide + ATP  P-Ser + ADP
o this catalytic enzyme has two domains, linked by a flexible region; each domain has a
specialized region with specific amino acid composition (small domain, large domain)
o glycine lid: a glycine-rich region, specifically used to block, bind and trap the nucleotide
(ATP); charged residues in the enzyme’s pocket stabilize phosphate groups of ATP
o active site: region between the two subunits; forms the groove where ATP will be localized
o the kinase has a surface devoted to recognizing a specific peptide sequence; it phosphorylates
the particular sequence, ie serine
o specificity of recognition site is distinct from that of enzymatic site: in order for enzyme to
function it is crucial for there to be 2 arginines on the N-terminal side (determines enzyme’s
specificity for its substrate); the 2 arginines and Serine bind to hollow groove
o proteins are dynamic: each step in reaction induces change in both substrate & enzyme
o open enzyme complex allows ATP to enter and substrate to bind  binding induces change in
conformation in enzyme, closing it  peptide + ATP brought in close proximity  reaction
where ATP is hydrolyzed and phosphopeptide is formed  products cause reopening of
enzyme, thus releasing product
o enzymes delocalize electrons to break the strong covalent bond (lysine and positive ions lock
the molecule and attract e-, which destabilizes the molecule)
Evolution of Multifunctional Enzymes
- reactions in cells usually occur as chain reactions, not as single reactions; to increase chain reaction
efficiency  scaffold protein
- scaffold protein: enzymes are grouped together and stabilized by scaffold protein; scaffold protein
only have protein-protein interaction domains, and so can recruit all components required for a
reaction to take place; also localizes reaction
- example: MAP-kinase pathway: an enzyme is activated that phosphorylates a kinase, which
phosphorylates another kinase and so on; MAP-kinase-kinase-kinase phosphorylates MAP-kinasekinase activates Map-kinase
Molecular Motors: Linear Motors + Rotary Motors
- proteins can be motors: actin and myosin motor in which myosin slides filaments, producing work;
protein motor causes bacteria flagellum to rotate
- a motor is something that displaces an object relative to another object: DNA and RNA polymerases
or ribosomes are motors because they produce force and displacement
- ATP synthase: enzyme that produces ATP in mitochondria; the complex machine acts like a motor;
uses proton gradient to synthesize ATP from ADP; enzyme has axis that turns ATPase subunits, this
rotation is required for ATP synthesis
o ATP synthase has 12 rotating “A” subunits, with identical composition, and a large subunit
with 2 channels for protons to travel through
o An acidic residue (aspartate) must be exposed at surface of each “A” subunit
o Proton (+) is attracted to aspartate (-) and neutralizes its charged; this changes the
conformation of that particular A subunit; in order to stabilize itself, it turns slightly to the
side
o Process is repeated on each individual residue, in each subunit; thus protons force the subunits
to rotate
Lecture 27: Regulation of Protein Function
-
regulation by changes in conformation: allostery and cooperativity
common regulators: calcium, GTP, phosphorylation/dephosphorylation
Allostery
- Allostery: binding of one ligand influences binding of another ligand in a different region of the
protein; because a protein is a network of intereactions involving covalent bonds of backbone and
non-covalent bonds between various amino acids, change implies formation of new non-covalent
bonds
- Positive allostery: binding of one ligand induces change in conformation of protein, which favors
binding of second ligand
- Negative allostery: modification decreases affinity of second ligand
Cooperativity
- cooperativity: binding of the first ligand induces changes in conformation that influence binding of B,
but alters conformation of the second subunit, favoring binding of the second A (first binding induces
allostery, involves several binding sites for the same ligand); cooperativity can also be negative
- cooperativity makes the system more sensitive to small changes in concentration of ligand
Allostery and Cooperativity: Regulation of Protein Kinase A
-
the catalytic part of PKA is made of two domains, which phosphorylates particular peptides;
regulated by pseudo-substrate that binds strongly to the catalytic site and cannot get phosphorylated
to release pseudo-substrate, bind cAMP; there are two binding sites per regulatory unit, so binding of
cAMP induces allostery that results in release of catalytic sites of the two regulatory units;
biochemically, inhibition of inhibition
ligand can now bind; binding of one ligand makes binding of second ligand easier
Cooperativity of Oxygen binding to Hemoglobin
- hemoglobin is a tetramer that binds 4 O2 molecules; interaction between hemoglobin subunits favors
binding of the next oxygen molecule
Calmodulin: Calcium-dependent regulator
- calmodulin: modifies activity of proteins; regulation dependent on calcium
- contains 4 calcium binding domains in protein; once activated (loop form), this protein interacts with
other peptides (globular) and modify their activity
- trick of regulation: Kd is low, and a small increase in low conc. of calcium in the cell is sufficient in
activating calmodulin loop
- calcium is at a low concentration in the cell because cell pumps out calcium as a messenger; calcium
concentration is higher in blood
Cycling GTP/GDP as a Common Switch Mechanism
- GTPase: series of enzyme that hydrolyzes GTP to regulate itself in order to regulate other proteins in
the cell; lousy enzyme because of slow hydrolysis and exchange
- Thus GEFs, GTP exchange factors, activate GDP  GTP
- GAPs, RGS, and GDI hydrolyze GTP  GDP
- Example: trimeric G protein has an alpha unit, which is a GTPase
o Interact with receptors sensible to hormones or light, changing the subunit to its active form;
causes dissociation of alpha subunit with beta and gamma subunits; the new freed surfaces
can now interact with target proteins
Phosphorylation: another Common Switch Mechanism
- phosphorylation: reversible cycle of an amino acid with a phosphate (kinase, active form) or no
phosphate (phosphatase); very dynamic
- example: receptor kinase: once phosphorylated, receptor kinase can bind adaptor proteins, and this
triggers cascade
Other Common Mechanisms
- cleavage: activation or inactivation; proteases are destructive machines, can be very specific; cleave
at specific sites that give a function to the products; ie insulin (proinsulin is cut at 2 sites by protease;
the two final fragments, forming insulin, are linked by disulfide bonds)
- subcellular location: the cell has compartments; some interactions occur at a specific location, so
activity can very from location to location; ie membrane anchoring and nuclear/cytoplasmic
localization of factors regulating transcription (modified by signals)
Introduction to Signaling Pathways
- cells respond to external and internal stimuli (growth factors, hormones, ions, extracellular substrate,
mechanical stress, other cells etc); binding of ligands to receptors trigger cascades in the cytoplasm
(network of interactions)
- signaling cascades regulate metabolism, ion channels, cytoskeleton, and nuclear gene expression
-
intracellular signaling cascades: protein-protein interactions, GTPases, Phosphorylation, proteolytic
cleavage, regulation of protein stability etc
gene activation/ repression via regulation of transcription factors
Lecture 28: Purification, Detection, and Characterization of Proteins I
Protein Purification
- protein are much more diverse than DNA, thus several methods are combined for purification
- separation of proteins according to physical and chemical criteria: mass (and shape), density, charge,
binding affinity
- 3 techniques in protein purification: centrifugation, electrophoresis, chromatography
Centrifugation: Mass and Density
- differential centrifugation: separation according to mass (=size)
o sample is poured into tube  centrifuge; particles settle according to mass  stop centrifuge
and decant liquid into container
o obtain 2 fractions: pellet and supernatant; crude method for particles of largely differing sizes
o separation optimized by changing centrifugation time and speed to separate bigger or smaller
fragments
- rate-zonal centrifugation: separation according to size + density
o sample is layered on top of gradient (increasing dense medium, such as sucrose; viscous
medium = slower sedimentation)
o centrifuge: particles settle according to mass; thus separation by size is better
o flaw: if some particles are less dense than the medium they will stop and float
- equilibrium density-gradient centrifugation: separation by density
o if rate-zonal centrifugation is spinned long enough and the density of the medium is high
enough, all the particles will eventually settle to density equal to themselves
o density gradient yields the purest DNA, since DNA is very dense
Eletrophoresis: Charge to Mass Ratio
- DNA is negative, attracted towards the cathode (+); thus separation is straightforward
- Proteins have variable composition, charge results from addition of all the charges of the protein;
most proteins have a negative charge and goes toward the cathode
- Modify pH to deprotonate proteins; negative charge moves faster, less mass moves faster
- This method retains protein structure; proteins are separated in this native state, properly folded; can
be retrieved to test enzymatic activity via assays
SDS-Page: Mass (Size)
- denature proteins completely with a strong detergent, SDS; now shape has no influence
- SDS: negatively charged detergent that binds hydrophobic residues of the polypeptide (which is
inside the structure), causing its unfolding + denaturation  all proteins become negatively charged
- Place protein mixture on cross-linked polyacrylamide gel and apply electric field; stain to visualize
the separated bonds
- Protein activity and protein-protein interactions is lost; however, gain high resolution
Isoelectric Focusing: Charge
- isoelectric point: sum of all charges = 0; depends on amino acid composition of each protein;
isoelectric point is expressed as pH, and different for every protein
-
pH gradient is established using special buffers (ampholytes) immobilized in acrylamide gel; proteins
are subjected to electric field and migrate, lose charge, and reach equilibrium line
two-dimensional gel electrophoresis: isoelectric focusing is often followed by SDS-Page: separate in
first dimension by charge  apply first gel on top of second  separate second dimension by size
Liquid Chromatography
- liquid chromatography: useful for isolation of large amounts of proteins
- principle: columns filled with polymer beads slow down all proteins down their path; size of
meshwork within the beads is calculated in a way that proteins have the probability to go into the
beads or bypass the beads
- based on size/mass (gel filtration), charge (ion exchange), and binding affinity
Gel Filtration Chromatography
- separation according to size (mass); analytical technique for small amount of proteins
- layer sample on column, add buffer to wash proteins through column, collect fractions
- tube is filled with polymer meshwork of beads; breads slow down molecules because path of particles
become very long; beads trap proteins that are small enough; larger protein = faster elusion
- any protein bigger than the pore size of polymer breads will not be separated; separation comes with
smaller proteins
Ion Exchange Chromatography
- separates proteins according to charge
- anion exchange: beads bind to negatively charged proteins; also cation exchange
- layer sample on column, positively charged gel bead collects negatively charged protein; elute
negatively charged protein with salt solution (NaCl)
- elution with NaCl: low salt = all negatively charged particles bind; medium salt = weakly charged
proteins are eluted; high salt = highly charged proteins are eluted
- charge of protein depends on pH, so binding of protein can be influenced by change in buffer pH
Antibody Affinity Chromatography
- load protein in pH 7 buffer; antibody beads; protein recognized by antibody stays on beads, protein
not recognized by antibody filters through; elute with pH 4 buffer to collect protein recognized by
antibody
- antibody-antigen interaction is pH sensitive  destabilized at acidic pH
- very effective purification, but never to purity
Protein Detection: Western Blotting
- to recognize protein of interest, transfer molecule from SDS-polyacrylamide gel (denatured protein)
to membrane, using electric current to force a faster transfer
- if protein has enzymatic activity, perform an enzymatic assay for detection
- antibody detection: incubate membrane with primary antibody for specificity; then incubate
membrane with enzyme linked secondary antibody for detection
- chromogenic detection: react with substrate for secondary antibody-linked enzyme
- Western Blotting provides antibody-antigen reaction AND size of protein (from gel electrophoresis
SDS-PAGE)
Lecture 29: Purification, Detection, and Characterization of Proteins II
Protein Sequencing: characterization of proteins
- edman method/high pressure liquid chromatography: choose peptide, label N-terminal amino acid,
then cleave N-terminal amino acid; identify aa by high-pressure liquid chromatography
o only sequences the first few amino acids; once a piece of the sequence is known, it can be
used to search in DNA sequence databases to determine entire sequence
- mass spectroscopy: bombard molecule with strong energy to break molecular bonds
o free radicals are produced (charged) and attracted to electrical field
o positively charged molecules are attracted to the detector; thus to get a positively charged
peptide, trypsin (a protease) is used to produce positively charged peptide, since it usually
cuts the fragment after reaching an Arginine, or Lysine, which are positively charged basic
amino acids
o magnet attracts free radicals according to mass; measures atomic mass at precise units (1 mass
unit)
o one can tune energy of laser to weaken peptide bonds of amino acids, producing peptide
fragments; each amino acid has a different molecular weight (except leucine and isoleucine),
thus one can identify a protein or a mixture of proteins
Determination of 3D Structure
- x-ray crystallography, cryoelectron microscopy, nuclear magnetic resonance NMR spectroscopy
- x-ray crystallography: most precise technique to determine structure of protein
o principle: when a beam of x-ray passes through a protein crystal, the electrons in the crystal
scatter the x-ray and produce a diffraction pattern of discrete spots on the detector film; by
analyzing the pattern produced, one can construct 3D structure of that protein
o limitation: since only crystal can produce diffraction pattern in x-ray crystallography, protein
must be crystallized; however, it is impossible to crystallize most membrane proteins, ie those
containing transmembrane domains
- NMR spectroscopy
o Principle: protein solution is placed in strong magnetic field; measure the influence of
neighboring atoms on the spin of atoms (generally H and C); frequency + strength of
magnetic field influences spin state of atoms
o Advantage: does not require protein crystal, more physiological
o Limitation: can only be used to analyze polypeptides that have less than 200 amino acids;
used to determine structure of protein domains
- cryoelectron microscopy
o principle: protein is rapidly frozen to preserve its structure; pictures of this protein are take at
different angles, and computer can reconstruct structures of this proteins by analyzing its
pictures
Proteomics: Comprehensive View of Proteins
- applications of proteomics
o isolate large protein complexes and assemblies and determine their composition
o isolate organelles and identify their components
o can also be used for known proteins: compare same protein from different cell type, determine
cell type-specific patterns of proteins or protein modifications; comparison of different
conditions, treatments (ie drugs)
- genomics can only reveal whether a gene is expressed or not, but will not show what modifications
has been done on that gene
Lecture 28B: Example of experiment using various methods: siRNA, cell fractionation, Western blot
and immunofluorescence
- role of RanBP3 in β-catenin nuclear export: what happens when RanBP3 is depleted?
Role of RanBP3 in β-catenin nuclear export
- RanBP3: Ran binding protein
- β-catenin: short half-life; undergoes ubiquitination; is a co-repressor or co-activator of gene
expression
- Wnt pathway: Wnt binds to receptors on the plasma membrane and triggers a signaling cascade
- Usually, β-catenin is in the cytoplasm and is degraded rapidly due to its short half-life; when Wnt
pathway is activated, β-catenin ubiquitination is blocked and b-catenin is stabilized; stabilized βcatenin is imported into the nucleus
- Without Wnt (hormone that triggers Wnt pathway), β-catenin is inactive (phosphorylated) and is
degraded rapidly; Wnt signaling pathway stimulates dephosphorylation of β-catenin, which permits
β-catenin to enter the nucleus, interact with transcription factors, and regulate transcription
- The goal is to test whether there is a shuttling mechanism and whether protein RanBP3 is involved in
export from nucleus
- A protein stabilizes β-catenin in the cytosol, but it shuttles; when we block export, the protein is now
stuck in the nucleus and the β-catenin left in the cytosol is degraded
siRNA
- to test RanBP3, the method of choice to partially get rid of the studied protein is to use siRNA, by
making a short plasmid transcribing a short precursor of the dsRNA with a loop
- when writing a sequence, only write one strand of the dsRNA in direction of transcription, so that the
sequence is equivalent of what will be the RNA sequence
Preparation of RNA interference
- search RanBP3 sequence in database, design 3-5 short interfering sequences
- synthesize two complementary oligonucleotides covering these short sequences, including flanking
overhands corresponding to restriction sites in vector; anneal the two oligonucleotides to produce a
dsDNA insert
- use a special vector optimized for expression of short hairpin RNAs: cut vector at two restriction sites
in polylinker, ligate insert; transform bacteria, pick clones, check presence of insert by restriction
digest, sequence region to verify right sequence
- transfect mammalian cells with newly constructed plasmids; verify that the clone is correct by
sequencing it
- test efficiency of RanBP3 depletion: transfect cells to test whether the plasmid has acted properly and
managed to deplete the protein properly
- western blot shows that sequences that target RanBP3 lead to its significant decrease; thus sequence
that targets RanBP3 is specific
Effect of RanBP3 depletion of β-catenin export
- in cells that are not treated with Wnt, no activated β-catenin Is present (all is phosphorylated)
- cells treated with Wnt hormone contain active β-catenin in the nucleus
- what happens when one activates β-catenin but removes RanBP3?
o Preparation of sample: break cells and biochemically separate nuclei (very large and heavy)
from the rest of the cytoplasm (soluble proteins) by differential centrifugation; supernatant
will be cytoplasm, pellet will be nuclei
o Analysis: analyze supernatant fraction with Western blot (with specific antibodies)
o for Wnt-treated cell that has lost RanBP3 after depletion, there is much more active β-catenin
in nucleus than in cytoplasm; thus RanBP3 plays a role in β-catenin export
Lecture 30: Protein Sorting/Targeting
-
a few proteins are synthesized in the mitochondria, while most are synthesized in the cytoplasm; they
have to know where to go
Cell Compartments
- mitochondria, nucleus, ER, Golgi, secretion, endocytosis all create compartments in the cell
surrounded by a biomembrane
- ER is connected to the nuclear membrane, forming a network
Two Major Pathways of Protein Targeting
- targeting to the membrane of an intracellular organelle
o occurs during or soon after synthesis of protein
o for membrane proteins, targeting leads to insertion in the organelle membrane; for watersoluble proteins, targeting leads to translocation of the protein to the interior of the organelle
o proteins go to the ER, mitochondria, chloroplasts, peroxisomes, and nucleus via this process
- targeting to the ER for subsequent secretion
o involves nascent proteins still being synthesized
o proteins are transported by small vesicles
- protein targeting involves signal sequences (20-50 aa segments that contain targeting information)
Experiment on Co-translational Translocation
- this historical experiment demonstrates that very soon after synthesis, proteins are already in the
lumen, inside vesicles
- pulse-labeling: a few radioactively-labeled amino acids are added only to newly synthesized proteins;
proteins are incubated in cells for a short period of time
- homogenization: cells put in a blender, vesicle is broken into little spheres called microsomes, which
still bind ribosomes
- conclusion: soon after synthesis, polypeptides can already be found inside vesicles
Co-translational Translocation of Secreted Proteins
- a signal sequence is present in all proteins targeted to the ER
- the ER signal is composed of charged amino acids, followed by a stretch of hydrophobic aa, almost at
the beginning
- ER signal is recognized and bound by a protein receptor: SRP (Signal Recognition Particle)
- Binding of SRP blocks translation
- SRP bind the SRP-receptor; thus SRP is a soluble protein that acts like a linker between the ER signal
and the SRP-receptor; the SRP-nascent peptide-ribosomes complex is now docked to the ER through
the SRP receptor
- The nascent polypeptide-ribosome complex is handed to a pore complex called translocon, which
opens; a little synthesized part of the protein is pushed in the ER lumen
- SRP is released, translation resues
- Once around 70 aa are translocated, a signal peptidase cleaves the signal sequence; the rest of the
polypeptide is translated and translocated
- Once synthesis is terminated, ribosomes leave and pore translocon closes
-
Energy is not really needed for translocation: translocation uses energy from translation, which
pushes the polypeptide chain through the pore
The only use of GTP hydrolysis in the SRP and SRP-receptor is for the purpose of control, like in the
case of chaperones
Translocation in Yeast
- in yeast, proteins are able to enter the ER after they have been synthesized
- translocation is unidirectional because BiP, a molecular chaperone found in the ER, is needed for
protein folding in the lumen; usually in the cytoplasm, Hsp70 takes care of folding; however, in this
system, chaperons in the lumen fold proteins because proteins are transferred directly from the
ribosomes to the ER
- the part of the polypeptide chain that crosses the translocon is bound by BiP chaperones, which have
affinity for the peptide and prevent it from sliding backwards
- even though Hsp70 is found in the cytosol, it doesn’t bind as strongly to the peptide as BiP; therefore,
BiP wins and the protein cannot slide back outside anymore; the more the peptide goes in, the more
BiPs bind
Targeting of Transmembrane Proteins
- many different scenarios apply for membrane proteins; unlike secreted proteins, which end up in the
lumen, transmembrane proteins are integrated in the membrane, halfway through synthesis
- a stop-transfer anchor sequence (hydrophobic transfer domain) is found in approximately the middle
of the protein
- when half of the protein is in the lumen and the translocon sees the transfer domain (signal), the
transfer domain is kicked out of the translocon and is integrated immediately in the membrane;
translocation is interrupted when the protein is no longer in the pore, and translation is completed in
the cytosol
-
one signal: once signal is cleaved, and the rest of the sequence has no transmembrane domain, the
protein will be free to float in the lumen
two signals: second signal acts as a stop sequence, can stop translocation and act as a
transmembreane domain
three signals: the third signal would be recognized as something to be integrated in the bilayer; thus
one can have alternated sequences
Targeting to Mitochondria
- mitochondria depend largely on nuclear genome for production of most of their components
- signal peptide for matrix proteins: 20-50 amino acid long amphipathic alpha helix (one side is
hydrophobic, the other is charged); the protein has to cross two membranes to get in there
- protein goes through translocons to get to the matrix; when a protein is translocated, its signal is
cleaved
- difference between ER and mitochodria: in mitochondria translocation, the protein has been
synthesized in the cytoplasm; it has to stay unfoled to go through the translocon
- proteins are maintained unfolded by constant binding of Hsp70; Hsp70 also brings protein to the
mitochondria
- if the protein wants to go into inner membrane or intermembrane space, it needs extra signals
- inner membrane protein: signal 1 = matrix signal (amphipathic alpha helix), signal 2 =
transmembrane domain that stops translocation so that protein stays in inner membrane
-
intermembrane space protein: signal 1 = matrix signal (amphipathic alpha helix), signal 2 =
transmembrane domain that stops translocation, signal 3 = cleavage site, so the protein is released
from the membrane and stays in intermembrane space
chloroplasts: similar to mitochondrial translocation