BI-334 Notes

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

Question 1: E, correct answer E

Question 2: E, correct answer E
Question 3: C, correct answer C
Topic 2: Chemistry in biology
Cells are mostly water and protein.
Molecular complementarity, requires both chemical and physical complementarity.
Exist this way (as numerous bonds) so that we have easier chemical reactions without require cleavage of covalent bonds.
Water is important (of course), because it’s polar, and hydrogen bonds are pretty darn important to proton pumping, by
working through a chain of hydrogen bonds.
Hydrogen bonds are all over the place in interactions, protein-protein, protein-RNA, etc
Protons can move through water molecules by attaching to one from another and them moving on in such a manner
Go and review O-Chem
The “Best” binding site is never used in biology because the best site forms too tight of a bond and cannot be broken and is
thus unusable
Go over page 40 again.
When covalent bonds are broken in a cell, it is almost always by enzyme, not always though, oxidative stress can also do it, for
example
Subunits covalently bond with each other allowing the structure to interact with itself via non-covalent bonds.
Go over the 20 amino acids and solidify their characteristics to learn where one might find them
Hydrophilic ones do the more interesting chemistry
Cysteine is used in disulfide bonds and glycine and proline are involved in protein folding
Most proteins are soluble in an aqueous environment and most will interact on the surface on a molecule
Basic
Lysine (K)
Arginine (R)
Acidic
Glutamate (E)
Aspartate (D)
Histidine can be charged or uncharged
Cysteine can make a covalent bond with another cysteine to change the way a protein folds, but this can only occur in the
endoplasmic reticulum
Don’t confuse nucleotides (have a phosphate) and nucleosides (don’t)
Know which are pyrimidines (CTU) and purines (A&G) and their structures

Question 4: B, correct answer B

Question 5: D, correct answer D
Question 6: E, correct answer E

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

Question 7: D, correct answer D

Question 8: C, correct answer C
Question 9: A, correct answer A

TOPIC 4

Motifs and domains are different

TOPIC 5
TOPIC 6
TOPIC 7
TOPIC 8
TOPIC 9
TOPIC 10

TOPIC 11

Have mechanisms in place to repair enough errors to maintain viability.

Humans average 3 mistakes for every cell division.
Most mutations are bad, which is to say the cells lose viability.
Mutations are critical to survival.
Semi-Conservative replication
Templated
Each strand of the double helix is copied.
Copies until we have two strands each of which has an old and a new strand.
Conservative Replication
Does not work. At all. Full stop.
Dispersive Replication
An interesting idea. But not possible in humans.
How to differentiate between strands
Meselson/Stahl Experiment proved semi-conservative replication.
How is DNA made?
What do we need to make DNA?
(2’)Deoxynucleotide triphosphates
Free 3’-OH <-DNA polymerase needs this and cannot make DNA without it.
Template
Enzyme
Add to the 3’ end
Pyrophosphate released
Replication fork
Asymmetrical as it has a leading stand, straight replication, and a lagging strand, where the okazaki fragments are
More than one polymerase at the fork.
Fidelity
Proofreading
Configuration of mispaired base detection
Deleted by 3’-5’ exonuclease
Replication restarted
Only works with 5’-3’ polymerization!
Replication Priming
DNA Primase with RNA polymerase activity with low fidelity
DNA Polymerase
RNAse H (Recognizes RNA/DNA hybrids and removes the RNA)
Primer Removal
RNAse H
DNA polymerase (fills in gap)
DNA Ligase (Seals both ends)
Opening up the DNA
Helicase opens up the double helix (depending on type they can open up going both directions
Protecting the exposed single stranded DNA
SSB or RPA (eukaryotes has RPA)
Binds single stranded DNA and does so without covering the bases.
Cooperative binding (one being bound increases likelihood of next being bound)
 Prevents hairpin formation
Processivity
Polymerase falls off – Processivity is a measure of how frequent it falls off.
DNA binding is rate limiting
Sliding Clamp
Processivity factor
Ring around DNA
Similar in all organisms (PCNA in humans, beta-clamp in bacteria, T4 in?)
Clamp Loader uses ATP to open up Sliding Clamp and then puts it around the DNA. Can identify 3’
DNA polymerase then binds to the sliding clamp.
Fixing mistakes-mismatch repair
MutS finds an error and MutL then locates a break and removes back to the mistake
How to prevent tangles
Supercoiling
Topoisomerase Topo I cuts and reseals a single strand. Topo II cuts double stranded before resealing both
Top I
Topo I
Tyrosine covalent bond attaches breaking
Top II Page 41
Creates a reversible covalent bond creating a double strand break forming a protein channel and then reseals the
break after the other helix passes through

Question 28: C, correct answer C

Question 29: D, correct answer D
Question 30: C, correct answer C

TOPIC 12

The Replisome is the complex at the Replication Fork

Know what’s on page 3
MCM complex named Mini-Chromosome-Maintenance gene
Steps of replication
Initiation
Origins – the region where replication starts
Initiator proteins determine location to start
RNA Primer put down
DNA Binding
Denaturation - split the strands
Recruitment
Elongation

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

Terminating sequences have directionality.

Tus system is not used in eukaryotic system
Histones after replication
H3-H4 tetramers are evenly distributed to both new strands intact by an unknown mechanism
H2A-H2B dimers are released after replication
Histone modifications after replication
Since each daughter nucleosomes only have the histones of the parent, reader-writer complexes recognize the
modifications are fill out the daughter chromosomes with the right histones.
Telomeres is made by telomerase
Used so that we don’t lose the ends of out DNA stands.
Makes a T-loop- the end loops back upon itself to create a structure that is resistant to nucleases
Telomere regulation
Long telomeres quickly shorten to normal
Same for short telomeres
Length is species specific.
Slide 44

Question 31: C, correct answer C

Question 32: B, correct answer B
Question 33: C, correct answer C

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.

Base excision repair

Base removed
Sugar phosphate removed
Repaired by DNA polymerase and DNA ligase
Nucleotide excision repair
Damage recognition
Incision in the damaged DNA strand on each side of the lesion
Excision of the oligonucleotide created by the incisions
Synthesis of new DNA to replace excised fragment
Ligation of the nick
Mismatch repair-replication errors
Occurs soon after DNA synthesis, requires identification of new strand
In E. Coli DNA methylation state is used, mechanism for recognition in eukaryotes is not known.
Double strand break repair
Non-homologous end-joining
Ku protein binds ends
Ku recruits DNA-protein kinase
Ligated by DNA ligase IV
Transcription coupled repair
Works on the template strand, other strand is repaired at the same rate as untranscribed DNA
RNA polymerase recruits enzymes involved in repair
Maybe focusing on the template strand because it’s the most important DNA
Homologous recombination
Another chromatid
 Process of broken strands
DNA deamination is easy detected
Photoreactivaton
Reverse pyrimidine dimers
Enzyme photolyases the butane ring
Emergency tactics: E.coli SOS response-cell arrest due to errors
Induced by cellular stress
RecA activated
Lots of genes expressed
Cell division stopped
Error prone polymerases expressed
Cell cycle block in eukaryotes-ATM

Question 37: D, correct answer D

Question 38: C, correct answer C
Question 39: A, correct answer A

TOPIC 14

When does recombination occur?

Meiosis it is critical for pairing of chromosomes which creates new chromosomes and diversity
DNA Damage repair, homologous chromosome is used
Replication restart-repair broken replication forks
In us it very rarely occurs in mitotic cells
Recombination
In crossover, a heteroduplex joint, where strands from two different DNA helices have base-paired off
Naturally occurring recombination
During meiosis
Mating type switching-yeast, inducible
B-Cell and T-Cell maturation
Lambda integration, site specific event
2 micron circle replication, naturally occurring extra-chromosomal piece of DNA in yeast, uses recombination for
replication
Transposons

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

RecA facilitates strand invasion

Starts as a single strand filament
Finds homology
Catalyzes strand exchange
DSB repair by homologous recombination

Two protein complexes facilitate

Holliday junction movement:
RuvA - tetramer
RuvB - two hexamers, one on either side of RuvA
Meiotic recombination
This process resembles double strand break repair, it starts with a double strand break
The enzymes that initiate the event are only expressed during meiosis
Holliday junction resolution

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

Question 40: C, correct answer C

Question 41: E, correct answer E
Question 42: E, correct answer B

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

Salmonella phase switching?

Phase variation is the alternation of expression of flagellar antigens H1 and H2 n salmonella typhimurium
It is mediated by site specific inversion of a 995 bp DNA segment of the chromosome
HIN, a protein encoded withing 995 bp segment, catalyzes the recombination between 14 bp inverted repeats flanking
the 995 bp segment called hix sites
SLIDE 22

Positive-negative selection enriches for targeted gene disruption

Question 43: X, correct answer X

Question 44: X, correct answer X
Question 45: X, correct answer X

TOPIC 16

Question X: X, correct answer X

Question X: X, correct answer X
Question X: X, correct answer X
TOPIC 17

Transcriptional termination in eukaryotes?

Pol II:
Very different
Does not involve secondary structure or rho factor
May not happen at all
RNA
Pol I:
Uses a terminator protein call transcription termination factor 1 or TTF-1 and RNA polymerase release factor
Requires two DNA elements: an upstream element that encodes 10-12 U residues and a downstream element
that binds TTF-1 called the Sal Box
Needs destabilization and a protein bound in the way
Pol III:
Appears to terminate in a cluster of four or more U residues
Not clear in any protein factors are required

How about Histones?

Question 49: C, correct answer C

Question 50: E, correct answer E
Question 51: B, correct answer B

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

Splicing removes introns from newly synthesized Pre-mRNAs

Percentage of gene that encodes exons varies
 Also, can have non-coding exons

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

snRNPs recognize the junctions via their RNA component

RNA rearrangements during splicing result in more accurate splicing
This not only allows catalytic activity but adds an additional check to make sure splicing is occurring at the correct site
Mechanisms to ensure splicing fidelity: why not make lots of mistakes?
Large size of introns make this more likely
In part, since splicing initiates immediately this is less of an issue since the entire RNA is not made yet
Exon size (length) is much more uniform that intron size
SR proteins help select exons
Assemble on exons to help mark the splice sites
These additional proteins may prevent the use of cryptic splice sites
They bind exons and proteins that recognize exons
Alternative spliceosome
Different snRNPs, similar mechanism
Trans!, uses SL snRNP, not in humans
5’ splice junction recognition
Takes advantage of splicing recognition mechanisms
Drosophila DSCAM gene, nervous system
38,016 possibilities – all functional
Mutations can change splicing site selection – remarkable plasticity
A single nucleotide change can
destroy a normal splice site, thereby causing exon skipping
destroy a normal splice site, thereby activating a cryptic splice site
creates a new splice site thereby causing a new exon to be incorporated

Poly A tails on mRNA

Consensus sequence for cleavage and poly-adenylation
Creation of the eukaryotic mRNA 3’ end SLIDE 35

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

only creates 1 precursor which is then modified and cleaved

modifications and cleavage positions are determined by special RNAs called guide RNAs that are members of a
class of RNAs called small nucleolar RNAs or snoRNAs

small nucleolar RNAs

SLIDE 46
Nucleolus
Not membrane bound, disappears n mitosis and then reestablishes
This occurs by the collective contribution of 10 chromosomes (in humans) where the rDNA repeats exist, looping out
DNA into a region to allow transcription of the rDNA genes
Where ribosome particles are assembled

Question 52: C, correct answer C

Question 53: D, correct answer D
Question 54: B, correct answer B

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

The ribosome is a ribozyme

RNA from the small subunit coordinates the codon/anti-codon interactions
rRNAs are responsible for both the structure and the catalytic activity of the ribosome
Ribosomal proteins stabalize the RNA core
Site selection is different between pro and eu
Polycistronic – Bacteria uses a recognition site called Shine-Delgarno
Monocistronic – scanning for AUG site
Prokaryotic translation initiation
IF2 promotes SLIDE 41

Question 55: C, correct answer C

Question 56: D, correct answer D
Question 57: A, correct answer A

TOPIC 20

Translation II
Eukaryotic Initiation & Post translational regulation

Eukaryotic translational start is not random

Bind mRNA and first tRNA are bound by small ribosomal subunit
initiator tRNAMet has unique factors that distinguish it from elongator tRNAMet
eIF4E (a complex) recognizes the cap structure on the mRNA
eIF4G (a complex) recognizes the poly A tail on the mRNA
the 5’ cap and poly A tail bind to eIF4 in a pseudocircle
Scan for AUG
This movement is mediated by other initiation factors that are ATP driven RNA helicases
 Not necessarily the first AUG, but the one that satisfies the Merilyn Kozak rules
Lose initiation factors and bind large subunit
Bind first aminoacyl-tRNA
regulate
Create first peptide bond
Other initiation
Another AUG can be used in addition to the initial one if a sequence called internal ribosome entry sites (IRES) exists
IRES folds into a structure that looks like a cap
Doesn’t need a cap but does require all the other initiation factors
Frequently found in viruses
Allows for a single RNA to code for two separate proteins in eukaryotes.
Translation Elongation
Bind new tRNA
Elongation EF-Tu is a GTPase that brings the charged tRNA to the A-site
Incorrectly paired tRNAs preferentially dissociate
GTP hydrolysis is thought to cause EF-Tu to release the tRNA which occurs if goog base-pairing exists
Form peptide bond
Translocate large subunit
Another elongation factor GTPase, EF-G, mediates mRNA and small subunit translocation
Translocate small subunit
Start again
Translation termination
Termination factors look like a ??? and binds to the stop site codon
Termination also requires GTP hydrolysis
SLIDES 21&22 important factors are highlited

Eukaryotic editing and quality control

Nonsense mediated mRNA decay
If stop codon is reached and exon junction complex is detected downsteam the mRNA is degraded. This is
triggered by the Upf complex
Upf triggers mRNA degradation
Long introns in mammals make this likely, the reason partial gene deletions result in null phenotypes
Prokaryotic quality control
Special molecule, tmRNA, with both tRNA and mRNA detects stalled ribosome
Attaches itself to stalled or broken mRNA and then codes for degradation
Template driven tag is recognized by proteases
Antibiotics affect ribosomes SLIDE 25

Post translational protein folding

Nascent polypeptide chain
Folding and cofactor binding
Covalent modification by glycosylation, phosphorylation, acetylation, etc
Binding to other protein subunits
Mature functional protein
Chaperones help fold proteins
Protein dynamics is also regulated by targeted proteolysis
The 26S proteasome degrades ubiquitinated proteins
A ring of ATPases thread proteins into the proteasome
Caps help with unfolding protein to feed through the central corridor full of proteases
The Ubiquitin pathway
Activation of ubiquitin ligase
Activated or triggered regulated
Activation of a degradation signal
 Must have site activation of some sort
SLIDE 36 sums up the central dogma

Question 58: B, correct answer B

Question 59: C, correct answer C
Question 60: D, correct answer D

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

Question 64: B, correct answer B

Question 65: D, correct answer D
Question 66: A, correct answer B

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

Are all repressor molecules in a cell bound?

How many does it take to activate transcription?
How can you turn off transcription when you are done?
How can you rapidly turn on transcription if necessary?

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

Eukaryotic Transcriptional Regulation is complex

RNA polymerase II transcription relies on proteins called general transcription factors which bind almost all pol II
promoters
In addition, there are several thousand gene regulatory proteins (8% of genes) that help provide specificity
These bind specific DNA sequences using DNA binding motifs we have discussed
Regulation more complex
Instead of a few cell signals, respond to dozens to dozens to affect gene expression
RNA polymerase II needs General Transcription Factors
Takes time to assemble the entire complex
No operons
All genes in a pathway must be coordinately regulated in the same way
SLIDE 6
Genes are regulated by hundreds of factors instead of a few

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

Insulators and Barriers regulate where enhancers work

Insulators and Barriers are both DNA elements
 Insulators block communication between an enhancer ad a promoter
Barriers prevent the spread of heterochromatin

Question 70: D, correct answer D

Question 71: B, correct answer B
Question 72: B, correct answer B

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

Question 73: E, correct answer E

Question 74: D, correct answer D
Question 75: C, correct answer C

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

In other examples of imprinting, methylation simply blocks gene expression by interfering

Imprinting SLIDE 15
Not known why it exists
Environment can control gene expression
Dosage compensation
Males are haploid for the X chromosome
Whole chromosome haploidy is usual lethal
There must be a mechanism to give balance of X (sex chromosome) products to A (autosomal chromosomal) products
in both sexes: dosage compensation
Whole chromosome inactivation
Males and females have different numbers of X chromosomes
To ensure the same amount of expression for both males and females one of the X chromosomes in females is
inactivated
X-chromosome inactivation SLIDE
This is called dosage compensation and maintains the X/autosome ration in males and females
The inactivation is random which means that females are genetic mosaics
SLIDE 20
Begins with synthesis of RNA from the XIC (X-inactivation center) locus
XICs on two X-chromosome sense each other and pair
Xist is transcribed on one X chromosome; Tsix on the other
Xist RNA coats the chromosome from which it is transcribed
SLIDE 22

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

Question 76: B, correct answer D

Question 77: B, correct answer B
Question 78: D, correct answer D

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

Question 79: D, correct answer D

Question 80: D, correct answer D
Question 81: B, correct answer B

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

Protein separation 1: liquid chromatography

Separation of proteins
Matrix (the material in the column), basis of separation
Ion exchange
Gel filtration
Affinity
Bind/retard
Hydrophobic interaction
Others
Collect fractions
Assay
Ion Exchange
Reversible, proteins can be eluted off
Medium resolution
Elution is achieved by slowly increasing the salt concentration
Gel filtration
Size exclusion
Low resolution
Very gentle
Like a sieve
Elution is continuous
Affinity chromatography
Very high resolution
>1000x purification
Elution varies, can use competing peptide
Used to purify
Antibodies
DNA
Ligands
Tagged proteins
His
FLAG
HA
MBP
Purification Steps: Ion exchange
Many proteins
OK resolution
some protein in the flow through
The rest is eluted with a salt gradient
Purification Steps: Gel filtration
Many proteins
Low resolution
Dilutes
 Gives size
Oligomeric state – monomer or dimer or what
Purification Steps: Affinity chromatography
High resolution
Sometimes final step
Can be very pure
Specific for protein of interest
SLIDE 21

Question 82: X, correct answer X

Question 83: X, correct answer X
Question 84: X, correct answer X

TOPIC 29

Question 85: X, correct answer X

Question 86: X, correct answer X
Question 87: X, correct answer X

TOPIC 30

Question 88: X, correct answer X

Question 89: X, correct answer X
Question 90: X, correct answer X