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Topic 1
Not microbiology, not related in the slightest. Also, not simply a technique, but a whole field.
Looking at the molecular mechanisms of processes. See slide, page ?
Cells make up all living things.
How is transcriptional regulation regulated.
Central Dogma: Replication -> transcription -> translation. Very important but plenty of exceptions.
Universal features of cells on earth.
All cells store hereditary information as DNA
Built from 4 nucleotides
Polar – directionality, one end is different from the other
DNA: Complementary, templated polymerization, redundancy
All cells replicate their hereditary information by templated polymerization.
RNA transcribed from DNA
RNA sometimes has folds with catalytic activity.
RNA synthesis called transcription
Amplification event: 1 DNA can lead to many strands of RNA and 1 RNA can lead to many proteins
All cells use proteins as catalysts.
Regulation very important and there are many mechanisms for it.
All cells translate RNA into proteins in the same way: ribosomes
Translation occurs in distinct steps
One gene = one protein
Genes are regulated
Various methods of regulation, internal and external regulation
Life requires free energy.
All cells function as biochemical factories dealing with the same basic molecular building blocks
All cells are enclosed by cell membranes which are selectively permeable
Life: an autocatalytic process.
Small molecules make polymers which can then go and regulate the small molecules
GENOMES
What is a genome?
The genome is the set of nucleic acids that specify the genetic information describing a given biological entity
ORF open reading frames
Two genes that do the same thing in different species are Orthologs
Two genes that do similar things are paralogs
MODEL ORGANISMS
Many aspects of biology are similar in most or all organisms
It is frequently much easier to study particular aspects in particular organisms
TOPIC 3
All about those enzymes, these are so important, though you’ve gone over this so many times your mind will wander
Don’t let it
You need to not get complacent
Most biological reactions are not energetically favorable and cells use energy to drive these necessary reactions
Enzymes can use the energy given by a energetically favorable reaction to drive an energetically unfavorable reaction
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Termination
Origin licensing
Prokaryote
Origin is fully methylated
There is a brief period where the origin is refractory, hemi-methylated origins are resistant to initiation.
Eukaryotic replication
Eukarotic DNA polymerases
Slow and replicates more DNA
Multiple origins of replication
ARS (yeast)
Temporally regulated
Cell Cycle regulation of DNA replication
Replication only occurs in S-phase
Euchromatic goes first and heterochromatic replicates last.
Autonomously Replicating Sequences (ARS) discovery
Discovered due to experiment with Yeast that cannot replicate without Histidine and the introduction of a plasmid that
has His gene and a random piece of yeast DNA.
Yeast Origins
~30,000 bp spacing
Eukaryotic origins are somewhat redundant as there are more than are needed. Thus, not all origins fire in every cell
cycle
Eukaryotic replication initiation – Slide 24
ORC is bound
Origin initiation
Origin is licensed once per cell cycle:cdc6
MCM is recruited (helicase), create pre replicative complex
G1-phase cdk activity causes cdc6 to be degraded (prevents MCM from loading again)
Initiation is then activated by S-phase cdks
DNA Pol alpha primes and then leading and lagging synthesis takes over (displacing the primase)
Note: Helicase moves on the template of the leading strand, in bacteria it moved on the template on the lagging
strand.
Human Origins of replication
How to prevent using an origin more than once per cell cycle?
Only G1 cells are competent to begin replication
S-phase cells contain a factor that starts DNA replication in G1 cells
G2 cells contain a block to their own DNA replication that cannot be activated by anything in the S-phase cells,
although this block is not soluble
What is this?
Licensing factor
TOPIC 13
DNA as a genetic molecule
All parts of DNA can be chemically modified
Chemistry of DNA is critical for information
Mutations can be inherited
Damage can come from virtually everywhere
Classes of point mutations – Slide 10
Transitions – AT and GC switching the locations of the purines and the locations of the pyrimidines
Transversions – AT to CG switching pyrimidines for purines and vice versa
Radiation damage
UV light – UV-A: very little damage, UV-B: responsible for most DNA damage in skin
Both involve dimer formation between adjacent pyrimidine rings on same strand -Slide 17
UV-induced cyclobutane pyrimidine dimer formation
6-4 photoproduct formation
Ionizing radiation
Direct damage – DNA or water bound tightly to it absorbs the radiation
Indirect damage – water or other molecules surrounding the DNA absorbs the radiation
Reactive species are formed that damage DNA
Double-stranded DNA breaks – thought to be the primary reason that ionizing radiation is so lethal to cells
Responsible for various chromosomal aberrations
deletions
duplications
inversions
translocations
Categories of DNA repair
Direct Repair – just fixes damaged nucleotides
Excision repair – excises damaged part and fills in using information on other strand
Mismatch repair – right after DNA synthesis
Nonhomologous end-joining – repairs serious damage, often causes mutations. Very important in antibody production
used to intentionally put in mutations.
TOPIC 14
Homology induced
During meiosis
During DNA repair
Site specific, catalyzed by enzymes called recombinases or integrases, works almost identically to topoisomerase II
HO
Flp
Rag 1&2
Lamda int
Cre, used today to make mouse knock outs of
Non-homology induced
Transposons
Parts of immunoglobulin production
Some DNA repair
Meotic recombination
Homologous chromosomes exchange material during meiosis in a process called recombination, recombination,
sometimes called crossing over or reciprocal exchange.
This process occurs essentially randomly across a chromosome so the frequency between two points (genes, or
markers) is related to the distance between those points (genes, or markers)
Genes that are far apart are more likely to be separated by recombination than adjacent genes.
How to recombine
What do we need?
Homology- base pairing
-100+ bp
Breaks
Strand invasion
Holliday junction
Branch migration
Resolution - will be enzymatically induced
Hybridization drives recombination
Double strand break – made by an enzyme or DNA damage
Processing
- 3’ single strand “tails”
Strand invasion – mediated by an enzyme
DNA synthesis
Branch migration
2 Holliday junctions
Resolution of Holliday Junctions
Gene conversion
Results in conversion of one allele to another, but started as normal homologous recombination evant
Thus, the allele now is present in three copies and the blue allele only exist as one copy
Called a 1:3 or 3:1 segregation event. The hallmark of gene conversion. Also call non-reciprocal exchange
Mating type switching results in gene conversion
“alpha” cells can switch to “a” cells via recombination
TOPIC 15
Transposons
DNA only transposons
Move by “cut and paste”
Transposase makes a staggered cut at the donor site for insertion
The gap left by ligating a blunt end to a staggered end is filled in by DNA polymerase
Repeats are created to either side of the transposed element
Retroviral-like retrotransposons
Move by “copy and paste”
Nonretroviral retrotransposons
Move by “copy and paste”
Repeats
Inverted -
Direct -
Life of a retrovirus
Entry into cell and loss of envelope
Reverse transcriptase makes DNA/RNA and then DNA/DNA double helix
Integration of DNA copy into host chromosome
Assembly of many ne infectious virus particles
Retrotransposons
Similar to retrovirus but lack coat proteins
Lots of repeated elements are transposon like
Non-retroviral retroelement replication
Reverse transcriptase
Endonuclease
Make a new copy every time!
Site specific recombination
Need
A DNA sequence that is specifically recognized, these sites are usually “directional” (not palindromic)
An enzyme or several that recognize the site and catalyzes recombination
Lambda integration
Integrase binds
Catalysis of Double-strand breakage and rejoining
Integrase dissociates
TOPIC 16
TOPIC 18
mRNA processing and splicing
Capping
Splicing
Poly-A(denylation)
RNA export
Nucleolus
Slide 3 quizing
Pre-mRNA much much bigger than mRNA
Prokaryotic mRNA can code multiple proteins
Eukaryotic mRNA for the most part only codes a single protein
RNA Polymerase II as an “RNA factory”
Phosphorylation creates a binding site
Tail contains 52 tandem repeats of a seven-amino-acid sequence
SLIDE 5
Capping proteins
Tail is phosphorylated
Released after transcribing
Creating a cap
Phosphotase
Guanyl transferase
Methyl transferase
Cap0: found in yeast
Cap1:
Cap2:
Capping enzymes recruited by CTD
Activated adenine acts as a nucleophile to attack another point to form a lariat and remove the intron from mRNA
Lariat as a unique 2’-5’ branch
Alternative splicing: many different proteins from the same gene
Specific sequences at the intron/exon junctions are required for splicing
The spliceosome
Splicing is mediated by 5 snRNAs
These are short (about 200bps) RNAs
Each one is complexed with at least seven proteins to form small ribonuclear proteins (snRNPs)
Together, along with 13 non-snRNP proteins, they constitute the spliceosome
After cleavage poly A polymerase (PAP) elongates the 3’ end by the addition of approximately 200 A nucleotides
No template
Added one at a time
As the poly A is being added it is bound by a protein called poly A binding protein (PABP)
Pol II transcription termination and poly A
Transcription termination requires poly A signals
Transcription termination requires factors for 3’ cleavage but does not require factors for polyadenylation
SLIDE 39
mRNA export
correctly processed mRNAs are exported through nuclear pores
this is an active process and thus requires energy
only mRNAs with the correct suite of proteins are exported SLIDE 43
other RNA processing SLIDE 44
TOPIC 19
Translation I
Understand the genetic code
Introduce tRNA
Understand how tRNA structure and function are related
Gain appreciation of the mechanism of tRNA activation
Learn how RNAs and proteins contribute to ribosome structure
Steps of translation
Initiation
Elongation
Termination
The Genetic Code
Instructions for assembling amino acids into proteins encoded into DNA
20 amino acids, only 4 bases
3 bases correspond to an amino acid
Most amino acids have multiple codons
In cases of more than one codon only the third base varies
Some have only one
Some codons are “punctuation”
The code is mostly universal
First two are more important that last one in a codon
ORFs
Start “AUG, GUG, UUG” always AUG in eukarya
Stop UGA, UAG, UAA
Only one reading frame encodes protein
tRNAs
How do we get from RNA to protein
For DNA to RNA we are able to base-pair and retain information
So we will need some sort of adaptor called a transfer RNA or tRNA
Usually 73-93 bp
Always base-paired
Can have some modified amino acids
Wobble hypothesis
More than one tRNA for each amino acid or “wobble” in the last position
Both! Varies by species
How does it work?
More than
The codon and anticodon form antiparallel base pairs
The first two bases in the codon from standard Watson-Crick base pairs with the last two bases in the anticodon
The third base of the codon and the first base of the anticodon can form certain nonstandard base pairs
How are tRNAs associated with amino acids?
Aminoacyl-tRNA synthetases
Most cells have 20 different ones (one for each amino acid)
What does that tell you about the specificity of the enzymes
Synthetase function
Binds to a specific tRNA and uses ATP to create a bond with
3 specificity sites: two amino acid binding sites and one tRNA binding site
The enzyme can determine in the amino acid is incorrect by determining if it fits into the editing site only the correct
amino acid is excluded
Method of protein synthesis.
Prokaryotes:
Coupled transcription and translation
Eukaryotes:
Uncoupled transcription and translation
RNA not associated with DNA
Ribosome parts
TOPIC 20
Translation II
Eukaryotic Initiation & Post translational regulation
TOPIC 21
RNA world
Theory
Substrates
Ribozymes
Prebiotic chemistry
Can result in complex molecules
Cannot accomplish heredity
Origins of life
Need to store information
Need to duplicate information
Need to be able to change information
Need to convert information into favorable reactions
Self-replicating element
RNA has most of the required properties
It can store information
It can template a copy of itself
It can change (mutations)
It can catalyze enzymatic reactions
However no RNA has been found in nature that is self-replicating, but at some point it had to have, right?
Pseudoknots
Found in
Ribozymes
Self slicing introns
telomerase
viral genomes*
*Many RNA viruses replicate by a rolling circle mechanism which results in concatamers
Mature genomes are created by a self cleaving event in which part of the genome folds into a pseudoknit releasing
genome sized RNA molecules
Ribozymes SLIDE 14
RNA can undergo conformational changes
Two folds for the same molecule have two different activities
Self ligation
Self cleavage
RNA synthesis by an RNA molecule
An ideal RNA world
Several functional RNAs cooperating
One replicates them
One protects them
One makes precursors
But they all need to be together! How?
Because greasy molecules tend to stick together
Question 61: C, correct answer C
Question 62: A, correct answer D
Question 63: D, correct answer D
TOPIC 22
Transcriptional regulation I: DNA binding
Concepts DNA binding
Gene expression critical for differentiation and signal transduction
Gene expression is regulated at many levels
DNA binding motifs
Are mostly Alpha helices in major groove
Are often dimers
DNA binding protein/site detection
Cell type specificity
Two completely different cells, differentiated
Same DNA
Some proteins the same, many different
Vastly different structure and function
A differentiated cell contains all the information to make an organism
Cell type specificity
How is this accomplished
mRNA expression patterns are characteristic of each cell type
protein expression is also characteristic of each cell type
Regulation of Gene expression
The six steps
Transcriptional control
RNA processing control
RNA transport and localization control
Translation control
mRNA degradation control
protein activity control
proteolysis
transcription factors
gene regulatory proteins
bind intact double standard DNA
sequence specific
ON or OFF
DNA can be read without disrupting base pair hydrogen bonding
The major groove displays unique patterns of H-bond donor and acceptor groups
DNA recognition regulatory regions are short DNA sequences – SLIDE 14
The DNA sequence recognized by a monomer does not contain sufficient information to be picked out from the
background of such sequences that would occur at random all over the genome.
For Example, an exact six-nucleotide DNA sequence would be expected to occur by chance approximately once every
4096 nucleotides. Clearly for a bacterial genome of 4.6x106 nucleotide pairs, not to mention a mammalian genome of
3x109 nucleotide pairs, this is insufficient information to accurately control the transcription of individual genes.
Therefore, many transcription regulators form dimers, with both monomers making nearly identical contacts with
DNA.
This arrangement doubles the length of the cis-regulatory sequence recognized and greatly increases both the affinity
and the specificity of transcription regulator binding.
Protein structural motifs found on gene regulatory proteins recognize DNA sequences
Common DNA binding motifs
Helix-turn-helix – mostly prokaryotic
Sequence specific binding-CRO repressor
Lambda CRO
Dyad symmetry – half sites-inverted repeats
Dimer
Homeodomains-special class of HTH domains
A special class of helix-turn-helix domains
A motif found in proteins that regulate development
Contains a third helix that all homeodomain proteins share, the third helix causes all the homeodomain HTH motifs to
be presented to DNA in the same way
Zn (Zinc) fingers
Multiple types
CxxC-HxxxH
CxxC-CxxC
Nuclear Hormone receptors and other transcription factors use this motif
Often exist as many fingers lined up in the major groove
Or function as dimers in which the spacing allows major groove contacts
This is a second type of zinc finger that has another helix and is similar to a HTH domain – It coordinates two zinc
atoms
Some DNA binding proteins have beta-sheets in the recognition domain
Some DNA binding proteins use loops that are not aha or beta structures
P53, for example, uses loops to interact with the DNA
Dimerization
Dimer Binding
Consensus sequence for transcription factor binding
Note: not that exact
Different factors can bind resulting in different binding modes and different types of regulation
– homo or heterodimers
Dimer binding-cooperative binding SLIDE 27
In some cases dimers can form in solution and then bind their site
– this results in non-cooperative binding
Often, the dimers only form after one binds in cooperative binding
Note that for cooperative binding you basically have either unbound or bound sites, in contrast non-cooperative
binding is characterized by a continuum of occupancy
Cooperative binding and histones
Dimerization motifs SLIDE 29
Dimerization is common and several protein domains are seen in DNA binding proteins that mediate dimer formation
Leucine zipper-coiled coil
Helix-loop-helix – similar, non-identical, proteins
Homo and Heterodimers
Mix and match
Combinatorial control
Vast increase in possibilities
Dimer regulation
Specific recognition of a base pair
Arginine in a very common side chain used to recognize guanine in a C-G base pair
Asparagine in in a very common side chain used to recognize guanine in a A-T base pair
Six similar zinc fingers binding different DNA sequences SLIDES 34-36
How to identify binding sites
Find a protein binding to a known site
EMSA
Electrophoretic gel Mobility Shift Assay
Band Shift Assay
Affinity chromatography
Take total cell proteins
Use salt washes to remove DNA binding proteins
Use specific washes to remove proteins that do not bind the specific proteins
Find a sequence bound by a known protein
DNA footprinting
Bind protein to DNA
Cleave DNA
Separate DNA
Footprint shows where protein was attached and thus where DNA could not be cleaved
Selex SLIDE 46
Systematic Evolution of Ligands by Exponential Enrichment
DNA binding protein site identification
Start with protein known
Large pool of a short DNA double helices each with a randomly generated nucleotide sequence
Separate protein-DNA complexes from free DNA using gel moilty shift assay
Remove protein and determine sequences of tight bound DNA fragments
ChIP SLIDE 48
Chromatin Immunoprecipitation
Cross link DNA-Protein
Break up DNA
Isolate protein
Determine bound DNA sequence
TOPIC 23
Transcriptional Regulation II: Bacteria
Transcriptional control – genetic switches SLIDE 2
Many genes coordinately expressed to accomplish mating type switch control in yeast
Using ChIP and footprinting, the entire set of genes that are activated or repressed during mating type switching was
identified
The tryptophan operon
Tryptophan production in E. coli
Operator: a unique regulatory sequence that can be bound by a protein to regulate RNA pol binding
This is called an operon: many genes involved in the same process, transcribed from a single promoter. First coined by
Jacob and Monod in 1961
Tre repressor requires tryptophan to bind the trp operator
When cellular levels of tryptophan are low, RNA polymerase binds the promoter and transcribes the genes
If the levels are high the repressor is activated and binds the promoter, preventing transcription
Tre repressor changes shape when bound to tryptophan
Binding to tryptophan changes the structure to correctly present the HTH domains to the DNA
Note: this is a dimer with HTH domains but the dimerization is not mediated by the HTH domains
The Trp operon has more tricks up its sleeve: attenuation
Attenuation
A unique regulator process
In E. coli only (bacteria)
Involves regulation of transcriptional termination by translation
The trp leader mRNA is translated and has two trp codons
Translation can affect transcription
The trp leader region can form alternative secondary structures
High trp causes attenuation and low trp allows transcription trp operon
High [trp], ribosome translates leader and RNAp terminates: No expression of trp operon
Low [trp], ribosome stalls in leader and RNAp continues: Expression of trp Operon
Nomenclature
Repressors turn genes off
Activators turn genes on
Ways to achieve positive and negative regulation SLIDE 13
Negative regulation
Bound repressor protein prevents transcription
Positive Regulation
Bound activator protein promotes transcription
Regulator proteins increase occupancy
Lambda SLIDE 15
Phage lambda has a complex regulatory region
The switch controls the decision between lytic and lysogenic growth
Repressor binds operator and determines transcriptional protein
Lambda repressor
Act as a repressor or an activator +/- 1 bp!
Depends on DNA binding site +/- 1 bp!
This dual role occurs in other systems as well. An example is CAP repressor
The Lac operon
The Lac operon encodes several genes
The lac operon includes cis-acting regulator elements and protein-coding structural coding genes
These genes allow cells to utilize lactose as sugar source
Expression of the lac operon in response to actose
Lac repressor binding overlaps the +1
The lac operon regulation: positive and negative regulators
Breaks down lactose
Expressed in absence of glucose and presence of (allo) lactose (a product of beta-galactosidase)
Here we have regulators functioning at once
One is sensitive to levels of glucose (CAP)
One is sensitive to levels of lactose (lac repressor)
Repressor binds as a tratramer
Helix-turn-helix
Inducer changes conformation of repressor (binding regulated by lactose)
Natural inducer is lactose
Catabolite repression SLIDE 28
CAP – catblite gene activator protein
Or
CRP – cAMP receptor prptein
Glucose down-regulates CAP (CRP) binding (an activator)
Similar to lac repressor except binding cofactor (cAMP) induces DNA binding
Galactose Operon
The gal operon is subject to both positive and negative regulation
Gal repressor binds as a dimer
Gl repressor and HU cause DNA looping and repression of the gal operon
araBAD operon
the araBad promoter region has multiple regulator elements
AraC undergoes a conformation change after binding arabinose
Action at a distance
Lac repressor binds several sites at once that are far apart
Increase likelihood of binding a second site after binding a first site
DNA loop size
Dramatically increases likelihood of two monomers binding if they are tethered
Since DNA cannot bend 90 degrees more than every 200 base pairs, a 100 base pair distance restricts the contact
between monomers
SLIDE 40-42
Alternative, interchangeable sigma factors
Viruses use sigma factors to activate transcription and progress through infectious cycle
rrn operons
the rrn operons contain both rRNA and tRNA genes
the IP element increases transcription of rrn operons
rRNA and tRNA are more abundant in faster growing E. coli cells
rRNA operons are subject to feedback regulation
Strategies for regulation of Gene expression
Operators
Dimerization
Multiple binding sites
Focus on mechanisms
Question 67: D, correct answer D
Question 68: E, correct answer B
Question 69: D, correct answer D
TOPIC 24
Transcriptional Regulation III: Eukarya
Eukaryotic Transcriptional Regulation is complex
Transcription factors are modular
Histones removed for activation
Repressors can act directly or indirectly
Protein-protein interactions are critical
In many eukaryotic processes, the interactions of multiple gene regulatory proteins are important for proper gene regulation
Utilizes Mediator to contact many gene regulatory proteins at the same time
Chromatin
Mediator interacts with activator proteins and Pol II transcription machinery SLIDE 11
These sequences that bind gene regulatory proteins are called enhancers
A cis-regulatory sequence that can elevate levels of transcription from an adjacent promoter
Many tissue specific enhancers can determine spatial patterns of gene expression
Enhancers can act
Properties of enhancers
Can act at a distance
Can be upstream or downstream of the transcription initiation site
Are orientation independent
Yeast upstream activating sequences are similar to enhancers in many respects but do not function when placed
downstream of the transcription initiation site
Gene activator protein are modular
DNA binding domain
Activation domains
SLIDE 17-21
Histone modifications influence transcriptional activity
Acetylation
Histone acetyltransferases (HATs)
Occurs on specific Lys residues in N-terminal tails
Enhances transcription by destabilizing nucleosomes
Deactylation
By histone deacetylases (HDACs)
Stabilizes compact chromatin structure
Tends to lower transcription
Chromatin and transcription
Polymerase and Mediator cannot displace histones to gain access to the DNA
How is this done?
All 4 of the histone modifying mechanisms we discussed already come into play
These are shown as separate pathways but they often work together in gene activation
Histone modifiers are recruited by gene activators
Gene activators can recruit histone modifying enzymes that in turn recruit remodeling complexes
Transcription activators can act at different steps SLIDE 27
Gene activation is synergistic, i.e. more than additive
Activation process
What order do these events occur?
Chromatin remodeling?
Binding in mediator?
Repressors of transcription
Eukaryotic transcriptional repressors can act in several ways
Direct – repressors binds at a location to prevent transcription
Indirect – SLIDE 32
Co-Activator/Co-repressor
Non-DNA binding protein
Weak protein-protein interactions – stronger than DNA
Some repressors and activators
The enhanceosome – SLIDE 34
Spatial transcriptional regulation
Developmental gene regulation
Secreted signaling protein gradients
Gene regulatory protein gradients
Complex regulation: beta-globin gen cluster SLIDE 48
Only expressed in red blood cells
Many activators, many repressors
TOPIC 25
Transcriptional Regulation IV: Regulatory circuits
SLIDE 2-3
Nuclear receptors are a large family of ligand dependent transcription factors
Transcription activation domain
DNA binding domain – binds near promoters to activate transcription
Ligand-binding domain – binds hormones and other small molecules and also mediates dimerization
A large number of them bind as heterodimers to the Retinoid X Receptor (RXR)
They bind similar DNA sequences; either direct or inverted repeats
Why mention NHRs?
These represent classic examples of:
The modular nature of transcriptional activators
The modular nature of binding sites
Ligand binding regulating transcription
Almost universal transcriptional regulations system
Also under regulation of coactivators and corepressors
Transcription regulated by heritable DNA rearrangements
Salmonella phase switching SLIDE 13-15
Yeast mating type switch
Cassette mechanism
HO endonuclease
Double-strand break induced gene conversion
Site specific recombination: mating type switching
Yeast mating type switching is initiated by the HO endonuclease
Yeast mating type switching transcription circuits
Regulatory Circuits
Circuit types – SLIDE 24
Positive feedback – a general strategy for cell “memory”
Negative feedback – way to keep critical levels of a protein constant
Flip-flop – like the lambda system to switch back and forth SLIDE 26
Consists of several positive feedback oops (combined to create a flipflop circuit)
2 stable states
Stable state 1: the prophage state, lambda repressor is made
Stable state 2: the lytic state, Lambda Cro protein is made
Feed forward – a filter to respond only to a certain type of signal (filter out short signals)
Simple gene clock
Drosophila circadian rhythm – A real clock
Coordination of gene expression at multiple sites
Glucocorticoid receptor
Binds hormone and can activate or repress transcription
Binds many other transcription factors to convert to high level expression
Affects different genes in different cell types (same signal)
Myogenesis (muscle) – a path for differentiation
Single transcription factor can lead to differentiation of muscle cells
Here, MyoD is expressed in fibroblasts (skin) and the cells make muscle
Differentiation-creation of many different cell types SLIDE 33
Many regulators in different cells at dfferent times
These different combinations result in different cell types
5 proteins
8 cell types
Differentiated cells can be converted into stem cells
When three specific transcription regulators are artificially expressed in cultured mouse fibroblasts, a number of cells
become induced pluripotent stem cells (iPS cells) – cells that look and behave ike the pluripotent ES cells that are
derived from embryos.
Summary
Permanent DNA changes can cause heritable changes in gene expression
Complex circuits of gene expression lead to different types of regulation
Some factors can turn on or off a large group of genes at once (glucocorticoid receptor)
Some factors can cause a cell to differentiate into a certain cell type (MyoD) and others
Some factors can cause the formation of an entire organ
TOPIC 26
Transcriptional Regulation V: Epigenetics
Epigenetics – changes in phenotype or gene expression caused by mechanisms other than changes in underlying DNA
sequence
Genomic imprinting
DNA methylation can lead to silencing but is not sufficient by itself
CpG islands are good ways to find genes
Whole chromosome inactivation
DNA methylation
5-methylcytosine
The only DNA methylation in eukaryotes
No change in base pairing
Only occurs in the sequence CG
Mechanism of DNA Methylation
The enzyme, maintenance methyltransferase, only methylates CG sequences based to methylated CG
Thus, methylation pattern is inherited
SLIDE 6
Roles for DNA methylation SLIDE 7
Methylation patterns can be used to establish heritable gene repression
In combination with other repression mechanism (that have been discussed) gene expression can be efficiently
repressed
Establishing DNA methylation SLIDE 8
CpG islands
Because of methylated C results in T, which is not recognized by the DNA repair system, CG dinucleotides are rare in
vertebrates
Those that remain are not evenly distributed but exist as CpG Islands – 1000-2000 nucleotides of CG
They are frequently found near Housekeeping gene promoters because they are not methylated
What are housekeeping genes?
LOOK THIS UP!
Why do CG islands exist? SLIDE 10
In germline cells, many genes are silenced
Mutations are mostly negative
Active gene regulatory regions are not methylated (by definition)
Genomic imprinting SLIDE 11
Some genes have only the paternal or maternal copy expressed, not both, this is called genomic imprinting
This phenomenon s caused by DNA methylation. This type of methylation is not removed after fertilization like
methylation
Thus, the pattern is inherited by somatic cells
Imprinting mechanisms
An insulator binds an element that prevents communication from the enhancer - thus, the gene is off
However, imprinting on the male-derived chromosomes results in methylation of the insulator binding site, preventing
insulator binding and turning on transcription
On the maternally derived chromosome SLIDE 13
Random
Not entire chromosome (why?)
A mechanism for dosage compensation, not the only mechanism seen in biology
Flies – Male X is hyperactivated – twice as much gene expression as each female X – whole chromosome
hyperactivation
Worms – Female Xs both down regulated so level of X products is the same in males and females - whole chromosome
hypoactivation
Humans – 1 X chromosome is (nearly) completely repressed - (nearly) whole chromosome inactivation by
condensation
Dosage Compensation complications
These regions are conserved and still have same functional genes and still cross over – pseudo autosomal regions
Pseudo-A regions do not dosage compensate
Whole chromosome inactivation
Does this only occur in sex chromosomes or can it occur (does it occur) in other chromosomes?
Yes!
Recent discovery-Human Molecular Genetics 2011 Apr 19 Thayer OHSU
Other examples as well-all seem to use Long Noncoding RNAs
Epigenetic Mechanisms
4 mechanisms for epigenetic inheritance
Epigenetic mechanisms that act in cis
DNA methylation
Histone modification
Epigenetic mechanisms that act in trans
Positive feedback loop by transcription regulation
Protein aggregation state
SLIDE 32
TOPIC 27
Post Transcriptional Regulation
Transcriptional attenuation and riboswitches
Alternative splicing
mRNA: localization and processing
RNA editing
Non-coding RNAs
Outline Post-transcriptional regulation
mRNA processing control
Attenuation and riboswitches
Alternative splicing
RNA editing
Translational control
Short non-coding RNAs
Long non-coding RNAs (IncRNA)
Post-transcriptional regulation pathways SLIDE 4
Attenuation (inducing termination event)
Alternative splicing
mRNA cleavage
RNA editing
Nuclear transport
Localization
Translational control
mRNA stability
Attenuation and the Trp operon
Riboswitch
A riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in
production of the proteins encoded by the mRNA
Attenuation and Riboswitches
Changes transcription termination hairpin loop dependent on Guanine concentration
Other examples cause ribosome stalling to prevent the hairpin from forming
Alternative splicing SLIDE 12
Exon skipping
Intron retention
Alternative 5’ splice site
Alternative 3’ splice site
Mutually exclusive exons
Alternative splicing: regulation
Alternative splicing can be passive
Due to sequence ambiguity, several possible events can occur at random
Many cases involve regulation and result in non-random splicing, this regulation can be positive or negative
SLIDE 14
Alternative mRNA cleavage in immunoglobulins
CstF – stimulates usage of a sub-optimal poly A site leads to a shorter transcript and a secreted form of the antibody
When activated to secrete, CstF is upregulated
RNA editing – addition of U nucleotides
Guide RNAs (encoded by the genome) direct sequence changes in the original message that add and remove uridine
nucleotides that alter the original reading frame and sequence.
Utilize an anchor sequence and U nucleotides are added by an enzyme called uridylyl transferase
Seen in the mitochondria of trypanosomes
SLIDE 20
Defense against viruses by denaturing their highly folded structures
Regulated nuclear transport
HIV genome
One long transcript makes 30 different mRNA molecules
Uses many different splicing patterns
Early in infection, a protein, Rev, is made and moved to the cytoplasm
Late in infection, Rev concentration increases and it enters the nucleus and binds to unspliced RNA and targeting it for
export
mRNA localization SLIDE 23-24
Translational control
Block Shine-Dalgarno or AUG
Translation repression from a ribosomal protein
Temperature regulates AUG access
Binding of a small molecule to a riboswitch that blocks SD sequence
An antisense RNA blocks the SD sequence
Global translational repression
Phosphorylation of eIF-2 – can occur under conditions of stress
Blocks recycling
Part of the mechanism for the establishment of G0 (quiescence)
Translational regulation
Leaky scanning
Sometimes first AUG not in a good region
Might go to next AUG
Can be used to make several versions of the same protein with different 5’ ends
Upstream ORFs
Sequester ribosomes
Prevent normal AUG from being used on the message
IRES
Folds into a distinct structure
Does not need Cap, but does need other translation initiation factors
Frequently found in viruses
mRNA stability
Bacteria
Mostly unstable – half lives of several minutes
Exonucleases responsible for this
Allows rapid response to environment
Eukaryotes
30 to 600 minute half lives
Endonucleases in some cases
Split leads to decapping of 5’ ends and rapid 5’-to-3’ degradation
Continued 3’-to-5’ degradation
5’ and 3’ ends both have methods of degradation
The same features are recognized for degradation and translation, thus they compete
Dual regulation: Aconitase – Fe regulation
Iron starvation
No Ferritin Made
Transferrin receptor made
Excess Iron
Ferritin Made
No transferrin receptor made
Positive
Stabilize mRNA
Negative
Repress Translation
Small non-coding RNAs
Another exception to the central dogma:
Non-coding RNA that regulates gene expression
Pol II transcripts
RNAs are destroyed in large complexes called P-bodies
Most regulate gene expression via an RNA interference mechanism (RNAi)
Small non-coding RNA that regulate gene expression
Three classes
microRNAs (miRNAs)
~22bp
small interfering RNAs (siRNAs)
piwi-interacting RNAs (piRNAs)
MicroRNA processing
Drosha releases dsRNA from microRNAs for dicer
Dicer releases dsRNA molecules for RISC (RNA-Induced Silencing Complex)
RISC consists of 4 proteins
AGO@, VIG, FXR, and Tudor-SN (TSN) assemble to form RISC
RISC protein functions – SLIDE 41
Ago2 – dsRNA helicase
VIG (Vasa intronic gene) – an RNA binding protein
dFXR or FmR - Fragile X
RISC
Unwinds dsRNA and uses the ssRNA to direct the complex to the RNa to be modified
RNA interference can cause chromatin structure to cause transcriptional repression SLIDE 43
This structure recruits chromatin remodeling factors that can lead to heterochromatin formation
RNA-Induced Transcriptional Silencing (RITS)
siRNA and DNA expressing shRNA can target the degradation of specific mRNAs in cultured cells
RNAi screens can explore the function of all genes in cultured cells
RNAi can be used to suppress genes in a tissue specific manner
RNA interference is a defense mechanism
Involved in defense against stranded RNA such as that is found in viruses and transposons
piRNAs SLIDE 48
A third system of RNA interference relies on PiRNAs (piwi-interacting RNAs , named for Piwi, a class of proteins related
to Argonaute).
piRNAs are made specifically in the Germ line, where they block movement
Bacteria use small noncoding RNAs to protect themselves from viruses SLIDE 49
CRISPR mediated immunity in bacteria and archaebacteria
After infection by a virus, a small bit of DNA from the viral genome is inserted into the CRISPR locus
A small fraction of infected cells must survive the initial viral infection and the surviving cells decendants, transcribe
the CRISPR locus and process the transcript into crRNAs
Long Non-coding RNAs SLIDE 50
Roles of Long noncoding RNA (lncRNA)
lncRNAs can serve as scaffolds, bringing together proteins that function in the same process
In addition to serving as scaffods, lncRNAs can,
STUDY SLIDE 51
TOPIC 28
Protein Methods
Centrifugation
Chromatography
Tags
SDS PAGE
Mass Spectrometry
Structural methods
BLAST
Outline Protein Methods SLIDE 3
Protein Purifications
Cell Fractionation-Centrifugation
Preparative
Sedimentive
Equilibrium
Protein Seperation-Chromatography
Ion exchange
Gel Filtration
Affinity
Protein Analysis
A problem
Cells are filled with thousands of proteins
How can we purify one to study what it does?
Need to open the cells, and separate the molecules
If done gently can retain integrity of structures
Centrifugation techniques separate particles that differ in mass or density
Cell fractionation 1-differential centrifugation
What moves faster, large or small things? Larger
Why is the centrifuge refrigerated? So that you don’t denature proteins with rotor heat and to keep enzymes from
activating.
What is lost when cells are burst open? Concentration. The contents are diluted.
Differential centrifugation
Low speed centrifugation,
supernatant subjected to medium-speed centrifugation
supernatant subjected to high-speed centrifugation
supernatant subjected to very-high speed centrifugation
Cell fractionation 2: sedimentation velocity centrifugation
A finer separation is achieved by layering the sample on top of a shallow gradient of a dense solution such as sucrose
or salt.
Separation is based on size and shape, and results in bands of similar material separating from others.
After spinning a set amount of time, the gradient can be dripped out the bottom to separate the components.
SLIDE 10
Extracts
These fractions can now be used to study various cell functions
Studies of purified organelles have revealed their cellular function
Studies of cytoskeletal components have shown details of important processes like mitosis, etc.
Others
Summary of centrifugation SLIDE 12
TOPIC 29
TOPIC 30