Cell bio chapter 8

cells differentiation is achieved by changes in gene expression because

differentiated cells express only half total genes in genome

leucine zipper

2 long helices coiled together, forming things that go into grooves to read DNA, leucine usually involved

2 alpha helices in bundle, leucine sticking out and bringing them together,

helix-turn-helix

first one found, in bacteria, tryptophan repressor is one of these, dimers helix with turn in between other helix

adds specificity one helix recognizes sequence of DNA other forms dimer

homeodomain

in higher eukaryotes, 3 helices, known for being in development, usually found in higher organisms, held with hydrophobic interactions,

has a recognition domain that is important for development in organisms

zinc fingers

lots of types, zinc coordinates beta sheets and alpha helix together, can hook together with other zinc fingers, reads DNA

very specific, can read 1000 bases

gene expression is the process by which

cells selectively direct synthesis or proteins and RNAs encoded in genome

genome from differentiated cells can be used to

direct development of complete organisms by injecting a nucleus into an egg without a nucleus can create a normal embryo

cells change expression of genes in response to

external signals, and cells can respond with different expression to same signals

transcriptional control is when and how

a gene is transcribed

rna processing control is how and

rna trnascript is spliced and processed

mrna transport and localization control

which rna is transported to nuclues

translational control is

which mrna are translated by ribosomes

rna degradation control is

how quickly mRNAs are degraded

protein degradation control is

how rapidly proteins are degraded

protein activity control

activity of individial proteins

promoter region include the

transcription initiation site (RNA synthesis begins) and upstream sequence (`50bp)

upstream sequences contain

recognition sites for sigma factor and general transcription factors

nearly all genes contain regulatory sequences are used to

switch on and off 10 nt-10,000 nt in length

regulatory sequences are bound by t

transcriptional regulators

binding transcriptional regulators to a regulatory DNA sequence can act as

a switch to control transcription

regulatory sequence bind in specific ways they

often inset into major groove of DNA double helix, contacts nucleotide pairs in groove without disrupting hydrogen bonds

four structural motifs of transcription regulators are

helix-turn-helix, homeodomain, leucine zipper, zinc finger

operons in bacteria and archaea act as

gene regulators

gene regulation allows for cells to

respond to environmental stimulation

tryptophan biosynthesis

operon is transcribed when tryptophan concentrations are low

operon is not transcribed when tryptophan is abundant

the tryptophan operon promoter contains an

operator sequence (regulatory sequence)

tryptophan operator is recognized by

transcriptional regulator (transcription repressor

the tryptophan regulator prevents transcription, and thus

tryptophan producing enzymes are also prevented when bound to operator

the tryptophan repressor is an allosteric protein meaning that

binding of tryptophan causes change in protein structure that allows it to bind to operator sequence

transcriptional protein in active form

switches genes off (represses)

transcriptional activator protein in active form

switches genes on (activates)

the genes that switch on and off with activators and repressors work on

promoters that bind rna polymerase poorly, often interact with second molecule to be able to bind DNA, bacterial activator protein CAP binds cAMP

the activity of a single promoter is often controlled by

two transcriptional regulators

the ecoli lac operon encodes proteins required for the

import and digestion of lactosel

ac repressor shuts off operon in the

absence of lactose

allolactose acts as the

inducer

allolactose is formed by

transglycosylation of lactose by b-galactosidase

CAP catabolite activator protein is an activator that

turns on gene expression in absence of glucose

cyclic AMP builds up when

glucose is scarce

enhancers are DNA sites where

gene activators bind in eukaryotes

silencers are DNA sites where

gene expression repressors bind in eukaryotes

the enhancers and silencers can be

a thousand nucleotides upstream or downstream of a gene

if these transcriptional regulators are so far away how are they able to regulate

DNA looping allows regulatory proteins to directly interact with the proteins at the promoter

proteins can serve as a link between

regulatory proteins and proteins at promoter

mediator complex aids in the

assemble of transcription complex at promoter

genes have regulatory regions that

dictate gene expression

in plants and animals DNA is in loops which allows for

genes and regulatory regions to be held in close proximity, localization restricts action of enhancers

groups of transcription regulators work together to determine expression of

a single gene known as combinatorial control

can activators and repressors be in the same complex

yes they can

cells need to coordinate gene expression of different genes because the

effect of a single transcriptional regulator can be decisive in switching particular gene on or off

a single gene can control many genes as long as

the same regulatory sequence is present

a given transcription regulator can control

the expression of hundreds or thousands of genes

genes are typically controlled by

many different transcription regulators

a set of three transcription regulators forms regulatory n

networks that specifies an embryonic stem cell

the formation of an entire organ can be triggered by

a single transcription factor

Ey controls the expression of multiple genes including transcription regulators, which can produce a cascade of regulators

acting as a master transciption regulatorsWork in combination to lead to formation of organized group of different cell types
Artificially induced expression of Ey gene can trigger formation of an eye

only a small number of transcription regulators may distinguish between cell types so the

induction of three nerve specific transcription regulators can convert a liver cell into a functional nerve cell

a combination of transcription regulators can induce a cell to de-differentiate for example

fibroblasts can be reprogrammed to become induced pluripotent stem cells

differentiated cells usually remain differentiated, so

proliferating cells maintain an identity(MEMORY)

the memory is because

patterns of gene expression are responsible for identity and must be remembered and passed on

positive feedback loop involves the

master transcriptional regulator activates the transcriptions of own gene in addition to cell-type specific gene

regulators are distributed to

both daughter cells, which continues to stimulate positive feedback loop

positive feedback is essential for establishing gene expression that allows cells to

commit to a fate and its state to its progeny

DNA methylation occurs on cytosines and it

turns off genes in vertebrates

methylation also

alters interactions with dna binding proteins and recruits proteins that block transcription

methylation patterns are passed to daughter cells by

copying pattern from parent strand dna to daughter strand by maintenance methyltransferase

variation in dna methylation are

responsible for imprinting

imprinting is the level of

gene dependent on parental origin

example of methylation

Mouse insulin-like growth factor 2 (Igf2)
Methylation of Igf2 gene on parental
chromosome leads to expression of gene
Mutation of maternal allele shows normal-
sized mouse (normal paternal allele
expressed)
Mutation of paternal allele shows dwarf
mouse (mutant paternal allele
expressed)

after DNA replication daughter chromosomes received half of parental chromosomes histone proteins which

contains covalent modifications present in parent, remaining stretched receive newly synthesized and (un-modified) histones

enzymes are responsible for each type of modification and they

create and catalyze spread of modification to new histones

mechanisms that transmit gene expression without altering the nucleotide sequence of dna can

work together to allow transient signals to be remembered by cell

alternative splicing allows for

different forms of proteins to come from the same gene, mRNA contains sequences that control how efficiently it is translated (translation initiation)

regulatory rnas are noncoding rnas that play and essential role in regulating gene expression, the major regulatory mrna are

micro rna mirna

small interfering rna sirna

long noncoding rna long ncrna

crispr rna

micro rnas base with specific

mrnas reducing the stability and translation of genes

micro rna regulate

1/3 of the proteins encoding genes (`1000 dif genes)

the micro rna precursor is processed to 22 nt by RNA dicer and it is packaged with

proteins to form rna induced stilencing complex RISC

the RISC complex searches for

mrnas complementary to the mirna to form base pairs, then the mrna is degraded by nuclease

a single micrna can inhibit the transcription of a

many different mrnas that contain a common sequence

cell defense mechanisms against double stranded RNA use some of the same components used with mirnas like

double strand being cut to 22nt by dicer, then RISC removes one strand of sirna duplex, searches for complementary strand rna is degraded by nuclease

the sirnas promote heterchromatin formation by

limiting the spread of transposable elements in the host genome

long noncoding rna are more than 200 nt long and estimation to be

5000 mcrnas in the human genome

lncrna x inactive specific transcript Xist is resonsible for

x inactivation 17,000 nt produced by only one x chromosome in nucleus, transcript coats chromosome promoting chromosome remodeling complex and modifying enzymes to for heterochromatin

some lncrna fold into

specific three dimensional structures by complementary base pairing, serves as scaffolds to bring together other proteins into complex RNA in telomerase

immune defense for bacteria and archaea

Organism that manages to survive a phage
attack captures a piece of invader’s
genome

goal: to block attempt of same type of phage to reinfect

Clustered regularly interspaced short
palindromic repeats (CRISPRs

consist of
repeats and spacers that do not encode
proteins
Spacer sequence between repeats correspond to
DNA from phage that infected cell

Near CRISPRs are CRISPR-associated (Cas)
genes that

do encode proteins (nucleases)
Nuclease proteins interact with CRISPR region

three stages of crispr functions

Adaptation - spacer acquisition
crRNA processing
Interference – targeting viral DNA/RNA

crispr and cas9

introduction of gene for Cas9 protein and
guide RNA can be used to study genome
function

genome engineering

Cas9, guide RNA, altered version of target gene
Homologous recombination replaces targeted
version of gene with altered version

gene activation/repression

iCas9, guide RNA, altered version of target gene
Homologous recombination replaces targeted
version of gene with altered version

pcr

amplifies DNA (usually specific genes)
Heat stable DNA polymerase (Thermus aquaticus; Taq polymerase)
Specific DNA primers (20-30 bases long) hybridize to known DNA sequences

pcr protocol

Heat 95 °C - separates (melts) DNA double helix
Cool to anneal primers, usually Tm minus 5 °C
Tm = melting temperature of primers; temperature that allows 50% of primers to bind DNA
Heat to 72 °C - optimum temperature for Taq polymerase

pcr should be repeated

25-30 cycles – each round doubles number of copies of DNA sequence

cells control

the activity and longevity of proteins
Controlling breakdown of proteins can regulate amount of protein in cell

Proteases enzymatically degrade proteins

Proteins whose lifetimes must be kept short
Recognize and remove damaged or misfolded
proteins

Eukaryotic cells use large protein machines:
proteasomes (present in both cytosol and nucleus)

Central cylinder from proteases
Active sites face central cylinder
Caps formed by large protein complexes
Bind proteins destined for degradation
Use ATP hydrolysis to unfold proteins
Thread them into inner chamber

ubiquitin is

small protein) covalently attached to proteins to be degraded
Specialized enzymes tag selected proteins with ubiquitin
Short lived proteins
Damaged or misfolded proteins
Oxidized proteins

Once proteins leave ribosome many require further
modification

Post-translational modifications:
Binding of small-molecule cofactors
Covalent modifications
Association with other protein subunits.