Block 4
Chapter 7
transcription/translation
Transcription: RNA polymerase uses one strand of DNA as template to synthesize a complimentary RNA sequence
takes place in nucleus
essential for protein synthesis. This process involves several key steps including initiation, elongation, and termination, which are regulated by various transcription factors and RNA processing events.
begins with the opening of the DNA helix to expose bases on each strand, one strand serving as template for RNA synthesis
ribonucleic acids are added one by one to the growing RNA chain, with each nucleotide being complementary to the DNA template strand.
RNA transcript is complementary to one strand of DNA and is synthesized in the 5' to 3' direction, ensuring that the genetic information is accurately copied for subsequent translation into proteins.
RNA chain is displaced and DNA helix reforms behind region where ribonucleotides are added, allowing for the separation of newly formed RNA from the DNA template, which ultimately stabilizes the overall structure of the double helix.
RNA molecule copied from limited region of DNA, making it shorter than the entire DNA strand, which enables specific genes to be expressed as needed during cellular processes.
Promoter: DNA sequence that initiates gene transcription, includes sequences recognized by RNA polymerase and its accessory proteins
binding of RNA polymerase to promoter opens up double helix
one strand acts as template for base pairing with ribonucleoside triphosphates
elongation continues until enzyme encounters second signal called terminator
DNA will be transcribed only if preceded by promoter
ensures only those portions of DNA molecule that contain gene will be transcribed to RNA
sigma factor (small unit of RNA polymerase) in bacteria responsible for recognizing promotor sequence on DNA-identifies sequence without having to separate entwined DNA strands because each base presents unique features to outside of double helix
Terminator (stop site): polymerase halts and releases both DNA tamplate and newly made RNA transcript
interaction of this 3’ segment of RNA with polymerases is what causes enzyme to let go of template DNA
Translation: Understanding how RNA sequences are converted into functional proteins, focusing on ribosomal activity and the role of transfer RNA (tRNA) in this essential cellular function.
before mRNA can be translated into protein, it must be transported out of nucleus through small pores in nuclear envelope and must go through RNA processing steps before exported to cytosol
sequence of nucleotides in an mRNA molecule directs incorporation of amino acids into proteins
genetic code: rules by which information contained in nucleotide sequence of gene and its corresponding RNA molecule is translated into amino acid sequence of a protein.
RNA polymerase
Enzyme that catalyzes synthesis of RNA from DNA template using ribonucleoside triphosphate precursors
catalyzes formation of phosphodiester bonds that link nucleotides together and form sugar-phosphate backbone of RNA chain
unwinds DNA helix, elongating RNA chain 5’-3’
RNA uses 3 types of polymerases for transcription
Polymerase I and III- transcribe genes encoding tRNA and rRNA
Polymerase II- transcribes the rest, including those that encode proteins
bacterial RNA polymerase relies on a single accessory protein, sigma factor, to initiate transcription
eukaryotic RNA polymerases require assistance of large set of proteins that arent subunits of RNA polymerase
ATP, CTP, UTP, GTP- ribonucleic triphosphates that provide energy needed to drive reaction forward
many RNA copies made from same gene in short time
can start RNA chain without primer and do NOT proofread work
general transcription factors
proteins that assemble on the promoters of eukaryotic genes near the start site of transcription and load the RNA polymerase in correct position
RNA polymerase II needs this
assemble on promoter- position RNA polymerase and pull apart double helix to expose template
process begins by binding of general transcription factors TFIID to short segment of DNA double helix composed primarily of T and A nucleotides- this part of promoter also known as TATA box
TBP and TFIID bind together at TATA box first then RNA polymerase II bind to initiate transcription. binding of TBP/TFIID slows movement of DNA
adding TFIIH and RNA polymerase II further slows TFIIH and RNA polymerase II alone won’t bind to DNA
once TFIID binds to TATA box, other factors assemble, along with RNA polymerase II, to form complete initiation complex
once RNA polymerase II positioned on promoter, must be released from complex of general trasncription factors to being task of making RNA molecule
key step-addition of phosphate groups to its tail-initiated bt GTF TFIIH-contains protein kinase as subunit
once transcription has begun, most general transcription factors dissociate from DNA and then available to initiate another round of transcription with new RNA polymerase
at the same time, new set of proteins aid in elongation loads onto actively transcribing RNA polymerase
elongation factors help polymerase move along DNA and gain access to DNA sequences that, in eukaryotes, are wound around histone proteins and packed into nucleosomes
If polymerase becomes dislodged from DNA before transcription complete, it must reinitiate the entire process from scratch at new promoter
only dephosphorylated form of RNA polymerase II can reinitiate RNA synthesis
Process Mechanics: Transcription Steps
Steps of Transcription:
Initiation: RNA Polymerase recognizes the promoter region and binds tightly, unwinding the DNA strand to access the template.
Initiation is critical because it is the main point at which cell selects which RNA to be produced
RNA must be able to recognize start of gene and bind to DNA at this site
Elongation: RNA polymerase synthesizes the RNA molecule complementary to the DNA template strand, moving along the DNA.
Termination: Transcription ends upon reaching a specific termination sequence; the newly synthesized RNA molecule is released for processing.
Template and Coding Strands: Distinction between template and coding strands is vital - the template strand serves as the guide for RNA synthesis, while the coding strand matches RNA nucleotide for nucleotide, save for U replacing T.
eukaryotes vs. prokaryotes transcription differences
Eukaryotic cells have several distinctive features:
Multiple RNA Polymerases: Each type of RNA (e.g., mRNA, rRNA, tRNA) is primarily synthesized by different RNA polymerases (e.g., RNA Polymerase II is chiefly responsible for mRNA synthesis).
Complex Initiation: Numerous transcription factors required for initiation of transcription show greater regulatory complexity in eukaryotes.
Chromatin Remodeling: Eukaryotic DNA is tightly wrapped around histones; transcription requires chromatin to relax to allow access to the DNA.
Complexity of Transcription Machinery: Eukaryotic transcription involves a broader array of proteins and factors, whereas prokaryotes rely mainly on RNA Polymerase and sigma factors.
RNA processing
Post-Transcriptional Modifications
takes place as RNA synthesized
enzymes responsible for RNA processing ride on phosphorylated tail of RNA polymerase II as it synthesizes an RNA, and process the transcript as it emerges from the polymerse
5' Capping: The addition of a methylated guanine cap to the 5' end of mRNA enhances stability and facilitates translation initiation.
modification of 5’ end of maturing RNA transcript - the part of mRNA that synthesizes first
in bacteria, 5’ end of mRNA is simply the first nucleotide of transcript
in eukaryotes, capping takes place after RNA polymerase II has produced about 25 nucleotides of RNA
3' Polyadenylation: Addition of a poly-A tail to the 3' end of mRNA increases stability and promotes nuclear export.
addition of multiple adenine nucleotides to 3’ end of newly synthesized mRNA
in bacteria, 3’ end of mRNA is end of chain synthesized by RNA polymerase
in eukaryotes, 3’ end first trimmed by enzyme that cutes RNA chain at particular sequence of nucleotides
transcript finished by second enzyme that adds a series of repeated adenine nucleotides to trimmed end (poly-A tail)
Capping and Polyadenylation mark the RNA molecule as mRNA
makes sure message is completed before protein synthesis begins
Splicing: Intron removal and exon joining enable variability in proteins through alternative splicing; this process is critical for producing multiple protein isoforms from a single gene.
intron sequences excised from RNA molecules in nucleus during formation of mature mRNA
introns removed and exons stitched together
each transcript receives poly-A tail
once transcript spliced and 5’ and 3’ ends modified, RNA is now a functional mRNA molecule that can leave nucleus and can be translated into protein
introns: noncoding sequence with a eukaryotic gene that is transcribed into RNA molecule but is then excised by RNA splicing to produce a mRNA
exons: segment of a eukaryotic gene that is transcribed into RNA and dictates the amino acid sequence of part of a protein
shorter than introns and represent a small fraction of total length of gene
Spliceosomes
Complexes of RNA and proteins that play a crucial role in splicing out introns. Spliceosomes consist of Small Nuclear RNAs (snRNAs) and proteins, collaborating to ensure mRNA maturation and functional efficacy.
large assembly of RNA and protein molecules that splice introns out of pre-mRNA in nucleus of eukaryotes
enzymes responsible for RNA processing ride on phosphorylate tail of RNA polymerase II as it synthesizes an RNA molecule so that the RNA transcript can be processed as its being synthesized
Chapter 8
gene expression
Gene Expression: The process by which a gene makes a product that is useful to the cell or organism by selectively directing the synthesis of a protein or RNA molecule with a characteristic activity
how different cell types turn on and off genes to achieve their specialized structures and functions
how transcription is regulated in prokaryotes and eukaryotes
Gene Expression can be studied by cataloging a cells RNA molecules, including the mRNAs that encode protein
involves determining the nucleotide sequence of RNAs made by the cell
Even within same cell type, gene expression can vary in response to external signals
Cortisol signals liver cells to boost production of glucose from amino acids. A eukaryotic cell can control when and how often a give gene is transcribed, how an RNA transcript is spliced and processed, which mRNAs are exported from the nucleus to cytosol, how quickly certain mRNA molecules are degraded, which mRNAs are translated to protein by ribosomes, how rapidly a protein is destroyed, and whether a protein is activated once it has been made
Transcriptional control can ensure that no unnecessary intermediates are synthesized. It is the regulation of transcription and the DNA and protein components that determine which genes a cell transcribes into RNA
Cortisol is a hormone released by adrenal gland in starvation, stress, exercise that signals to out liver to increase the production of glucose from amino acids-upregulation of gluconeogenesis genes
Cells can change which genes they express without altering their DNA sequence
Differentiations: process by which a cell undergoes a progressive, coordinated change to a more specialized cell type, brought about by large scale changes in gene expression
generating different cell types
different cells produce different proteins
how differentiated cells maintain their identity
housekeeping
expression of different collection of genes and production of specialized proteins making each cell different
Housekeeping proteins: RNA polymerases, DNA repair enzymes, ribosomal proteins. enzymes involved in glycolysis and other basic metabolic processes
Housekeeping genes: encode housekeeping proteins
Specialized cells are capable of altering their patterns of gene expression in response to extracellular cues
A neuron and liver cell contain the same genome but express different RNAs and proteins
techniques to compare protein composition between cells (mass-spectrometry and quantitative proteomics)
Sequencing RNA and mRNA molecules used to profile gene expression
Different cells of an organism vary not because they have different gene (they all have the same genes), but they express them differently
gene expression patterns depend on the context and signals
controlling gene expression
The expression of gene into protein controlled at many levels and multiple mechanisms are used to control it at each level
Genes can be turned on or off
For a gene to be expressed, it must first be transcribed by RNA polymerase which binds to the promoter sequence
assisted by transcription factors (eukaryotes)
Promoter: DNA sequence that initiates gene transcription; includes sequences recognized by RNA polymerase and its accessory proteins
binds to RNA polymerase and correctly orients enzyme to begin its task of making an RNA copy of gene
Genes include transcription initiation site, where RNA synthesis begins, plus nearby sequences that contain recognition sites for proteins that associate with RNA polymerase
In addition to promoter, nearly all genes have regulatory DNA sequences that regulate transcription
these sequences recognized by transcription regulators
Regulatory DNA sequences: DNA sequence to which a transcription regulator binds to determine when, where, and in what quantities a gene is to be transcribed into RNA
switch genes on and off
have to be recognized by transcription regulators
transcription factors/regulators
Proteins that can bind to a specific DNA sequence and regulate transcription and gene expression
General Transcription Factors (TFIID): help RNA polymerase II locate the promoter and initiate transcription (eukaryotes only, in prokaryotes the sigma factor recognizes the promoter)
Specific Transcription Factors: activators and repressors influence the efficiency or rate of transcription initiation by promoting or blocking the recruitment of the RNA polymerase to the promoter (eukaryotes and prokaryotes)
Transcription Regulators: protein that binds specifically to a regulatory DNA sequence to switch a gene on or off
what determines if a gene should be on or off
recognize specific nucleotide sequence to do so because the surface of the protein fits tightly against the surface features of the DNA double helix
because the surface features will vary depending on the nucleotide sequence, different DNA-binding proteins will recognize different nucleotide sequences
protein inserts into the major groove of DNA double helix and makes non-covalent contacts (form hydrogen bonds, ionic bonds, and hydrophobic interactions) with the nucleotide pairs within the grove
many transcription regulators bind to DNA helix as dimers
dimerization doubles the area of contact with DNA, increasing potential specificity and strength of the protein-DNA interactions, as well as increasing tightness of interaction
as long as different genes contain regulatory DNA sequences that are recognized by the same transcription regulator, they can be switched on or off together
ability to switch many different genes on or off using a limited number or transcription regulators is useful in regulation of cell function
some transcription regulators can be used to convert a differentiated cell into an entirely different cell type
single transcription regulator control the expression of multiple genes to direct cell differentiation
can also trigger the formation of an entire organ: Ey transcription regulator in fruit flies control the differentiation of cell that form eye
flies with mutation in Ey have no eyes
some of the genes regulated by Ey encode other transcription regulators that regulate the expression of other genes
Ey is an example of a master regulator that produces a cascade of regulators that work together to regulate the formation of the whole organ
artificially expressing Ey in precursor leg cells cause the formation of (non-functional) eye structure on the fly leg
can be used to direct the formation of specific cell types or convert one differentiated cell type to another
myoD is a muscle-specific transcriptional regulator
each cell type is defined by the combination of transcription factors it expresses
operon
clusters of genes that are transcribed together into single mRNA because they share the same promoter
regulated by repressors or activators
Genes in bacteria are arranged in a cluster on the chromosome and transcribed from a single promoter as one long mRNA molecule
called an operon: mostly found in bacteria, rare in eukaryotes
the tryptophan (trp) operon contains five genes, which encode five different enzymes needed to synthesize tryptophan
when tryptophan concentrations low, operon is transcribed/expressed; the resulting mRNA is translated to produce a full set of biosynthetic enzymes, which work to synthesize the amino acids from simple precursor molecules
within the operon’s promoter is a short DNA sequence called the operator, which is recognized by a transcription regulator
when regulator binds to operator, it blocks access of RNA polymerase to promoter, preventing transcription of the operon and production of the tryptophan repressor; the repressor can bind to DNA only if it also bound to tryptophan
tryptophan repressor is an allosteric protein
binding to tryptophan can cause a change in its 3D structure so that the protein can now bind tightly to operator sequence
when tryptophan concentrations high, operon expression is repressed
DNA-binding motifs
Transcription factors form tight non-covalent interactions with the DNA major groove
bind as dimers
A motif is a short and conserved amino acid sequence in a protein associated with a distinct function, but cannot function on its own
DNA-binding motifs
homeodomain motif
helix-turn-helix motif
zinc finger motif
leucine zipper motif
Homeodomain Motif
found in many DNA-binding proteins
involved in regulatory control of developmentally important genes across organisms
consists of three a helices (cylinders) which fit the protein to the DNA major groove
contacts with the DNA bases are made by helix 3
specific type of helix-turn-helix motif
Helix-turn-helix Motif
two adjacent a helices separated by turn of several amino acids
only one helix contacts the DNA bases in the major groove
the action of the homeodomain motif is mediated by this motif
Zinc Finger Motif
built from a helix and b sheet held together by molecule of zinc (helps stabilize structure)
often found in clusters to allow the a helix of each finger to contact the DNA bases in the major groove
Leucine Zipper Motif
bind to DNA as dimers gripping the double helix like clothespin or scissor-grip
formed by two a helices, each contributed by different protein
many transcription factors bind to DNA as dimers doubles the area of contact with DNA
increases specificity and strength
repressors/activators
Transcriptional Repressor: protein that binds to specific regulatory region of DNA to prevent transcription of an adjacent gene
switches gene off
Transcriptional Activator: protein that binds to a specific regulatory region of DNA to stimulate transcription of an adjacent gene
switches gene on; can bind to RNA polymerase on their own and can be made fully functional by activator proteins that bind to a nearby regulatory sequence
Lactose (Lac) operon: encodes proteins required to import and digest the disaccharide lactose
activated by catabolite activator protein (CAP)
binds to cyclic AMP (cAMP) before it can bind to DNA
in the absence of glucose, the bacterium makes cAMP, which activate CAP to switch on or activate genes that allow the bacteria to digest other sugars including lactose
low glucose level=cAMP levels increase
CAP binds to cAMP and activate expression of enzymes to metabolize lactose
If lactose isn’t present, the lactose repressor will shut down the expression of the Lac operon
Lac operon regulated by both activators and repressors depending on the sugar
integrates two different signals
1. glucose must be absent
2. lactose must be present
Lac repressor shuts off the operon in the absence of lactose
glucose must be absent and lactose must be present
The DNA sites to which eukaryotic gene activators bind are called enhancers
presence of enhancers dramatically enhances the rate or transcription
unlike bacteria, eukaryotic activator proteins can enhance transcription even when they are bound to thousands of nucleotide pairs
Lac regulatory components
includes three genes that encode three enzymes needed to import and digest lactose (if glucose is not available)
LacZ: the first gene of the operon, encodes the enzyme b-galactosidase, which breaks down lactose to galactose and glucose
CAP-binding site (Lac activator): RNA polymerase binding sit (promoter), operator (Lac repressor binding sit)
glucose and lactose concentrations control the initiation of the transcription of the Lac operon through their effects on the Lac repressor protein and activator protein CAP
Lac operon genes are normally transcribed when two conditions met
1. glucose is absent - activator binds to CAP-binding site
2. lactose is present - allolactose binds to the repressor and releases its grip on the operator DNA
High glucose - low cAMP - CAP cannot bind to CAP-binding site
High lactose - high allolactose - allolactose binds to Lac repressor - lac repressor dissociates from operator region
Both activator and repressor are NOT bound, and Lac operon is OFF
DNA packaging
nucleosomes are DNA wrapped around protein core
Histone: highly conserved proteins around which DNA wraps around to turn nucleosomes
responsible for the first and most fundamental level of chromatin packing; the formation of the nucleosome
histones and non-histone proteins enable DNA packaging
such packaging may have evolved to prevent “leaky” gene expression and allow for regulation
chromatin-remodeling proteins and enzymes covalently modify histones that form the core of nucleosomes
chromatin remodeling
use energy of ATP hydrolysis to alter the arrangement of nucleosomes in eukaryotic chromosomes, changing accessibility of the underlying DNA to other proteins
uses energy of ATP hydrolysis to change the position of nucleosomes on the DNA
transcription activators can recruit histone-modifying enzymes and chromatin-remodeling complexes to the promoter region, making chromatin more accessible to the transcription factors and RNA polymerase
Histone acetylases: add acetyl group to select lysine - makes DNA more accessible - enhance transcription efficiency
Histone deaceylases: remove an acetyl group and restores packaging
makes DNA less accessible
inactivates expression
gene activation
activators bind to enhancers - regulatory DNA sequences that enhance transcription and are located far away from the promoter regions
binding of activators to enhancers causes DNA to bend
this positions activators bound to the enhancers closer to the general transcription factors and RNA polymerase II
activators also interact with mediator
mediator: large complex of proteins that serve as adaptors to close the loop
all work together to form the transcription initiation complex
DNA loops
plant and animal chromosomes are arranged into loops - also called TADs: topological associated domains
specialized proteins hold/clamp loops together
range in size
positive feedback loop: important form of regulation in which the end product of a reaction or pathway stimulates continued production or activity; can control a variety of biological processes
production of protein may stimulate continued production which keeps cell from changing into another type of cell
generates cell memory
ex. MyoD directs the cells to become muscle cells
stem cells
when a stem cell divides, each daughter can either remain a stem cell or go on to become terminally differentiated
both stem cells and proliferating precursor cells are usually retained in their resident tissue along with their differentiated progeny
specific signals maintain stem-cell populations
stem cells can be used to repair lost or damaged tissues
differentiated cells
specialized cells can be induced in culture
Induced pluripotent stem (iPSs) cell: somatic cell that has been reprogrammed to resemble and behave like a pluripotent embryonic stem (ES) cell through the artificial introduction of a set or genes encoding particular transcription regulator
behave much like naturally occurring ES cells, and they can be directed to generate a variety of specialized differentiated cells
once a cell has become differentiated into a particular cell type in the body, it will generally remain differentiated and all its progeny cells will remain the same cell type
Terminally differentiated: never divide again once they have been differentiated (skeletal/neural)
Cell memory: ability of differentiated cells and their descendants to maintain their identity
DNA methylation: the enzymatic addition of methyl groups to cytosine bases in DNA; the covalent modification generally turns off genes by attracting proteins that block gene expression
passed on to progeny cells by an enzyme that copies the methylation pattern on the parent DNA strand to the daughter DNA strand as it is synthesized
Three ways a cell can maintain its identity
positive feedback loop: a master regulator activates its own transcription at every division thus generating a “self-sustaining” circuits of gene expression (most prevalent way)
condensed chromatin: by histone modification - histones are passed from parent to daughter cell (the same X chromosome in all mammalian females is inactive through many generations)
DNA methylation: covalent modification - methylation of cytosine, generally methylation turns OFF transcription
generally these are epigenetic changes- meaning that no changes in the DNA nucleotide sequence occurs
post-transcriptional controls
regulation of gene expression that occurs after transcription of the gene has begun
operate after RNA polymerase has bound to gene’s promoter and started to synthesize RNA
alternative RNA splicing and protein degradation
Regulatory RNAs: RNA molecule that plays a role in controlling gene expression
microRNAs, small interfering RNAs, CRISPR RNAs, and long noncoding RNAs
The endogenous “noncoding RNA” (precursor miRNA) is processed to form mature miRNA with the help of nuclease dicer
mature miRNA is packaged with specialized proteins to form a RISC (RNA-induced silencing complex), which patrols the cytosol in search of mRNAs that are complementary in sequence to its bound miRNA
once a target mRNA base-pairs with an miRNA, it is either destroyed immediately by a nuclease that is part of the RISC or its translation is blocked'
bound mRNA released the miRNA-bearing RISC, allowing it to seek out additional mRNA targets thus, a single mRNA as part of RISC, can eliminate one mRNA molecule after another efficiently blocking production of the encoded protein
mRNAs life span is dictated by specific nucleotide sequences within the untranslated regions that lie both upstream and downstream of a protein-coding sequence
sequences contain binding sites for proteins that are involved in RNA degradation and carry information specifying whether and how often the mRNA is translated into protein
Bacterial mRNAs contain a short ribosome-binding sequence located a few nucleotide pairs upstream of the AUG codon where translation begins
this binding sequence forms base pairs with the rRNA in the small ribosomal subunit, positioning the initiating AUG codon within the ribosome na provides an ideal target for translational control
by blocking or exposing the ribosome-binding sequence, the bacterium can either inhibit or promote the translation of an mRNA
In eukaryotes, specialized repressor proteins can similarly inhibit translation initiation by binding to specific nucleotide sequences in the 5’ untranslated region of an mRNA, preventing the ribosome from finding the first AUG
RNA interference (RNAi)
cellular mechanism is activated by double-stranded RNA molecules that results in the destruction of RNAs containing a similar nucleotide sequence, it is widely exploited as an experimental tool for preventing the expression of selected genes (silencing)
In the first step of RNAi, RNAs are cut into short fragments in the cytosol by a protein called dicer
dicer: the same protein used to generate the double-stranded RNA intermediate in the miRNA pathway. The resulting double-stranded RNA fragments called small interfering RNAs are then taken up by the same RISC proteins that carry miRNAs
RISC discards one strand of the siRNA duplex and uses the remaining single-stranded RNA to seek and destroy complementary RNA molecules
RNAi can selectively shut off the synthesis of foreign RNAs by the hosts RNA polymerase
siRNAs produced by dicer are packaged into a protein complex called RITS - attaches itself to complementary RNA sequences as they emerge from an actively transcribing RNA polymerase
RITS complex attracts proteins that modify histones resulting in heterochromatin formation that causes transcriptional repression
CRISPR: small noncoding RNA system that provides bacteria with immunity from viral infections
resembles RNA interference and makes use of small noncoding RNAs, similar to siRNAs as guides that direct the identification and destruction of invading viral genomes
when a virus injects its DNA into a bacterial cell, short fregments are removed from the viral DNA and inserted into CRISPR locus
bacterial cells use this information to produce a collection of short guide RNAs calls CRISPR RNAs which are individually loaded into a CRISPR associated enzyme called Cas.
Case then uses its bound RNA to seek out and destroy any complementary viral nucleotide sequence it might encounter
long noncoding RNAs
class of RNA molecules that does not encode proteins; often used to regulate gene expression
Xist (X-inactive specific transcript): key play in X inactivation- the process by which one of the two X chromosomes in the cells of female mammal is permanently silenced
essential for initiating and maintaining epigenetic silencing of one copy of X chromosome by promoting heterochromatin formation
the transcript then sticks around, coating that chromosome and attracting the enzymes and ATP-dependent chromatin-remodeling complexes that promote the formation of a highly condensed form of heterochromatin
small long noncoding RNAs fold into specific 3D structures via base-pairing and can serve as scaffolds; provide a template for telomere synthesis, the large RNA molecule that is part of telomerase holds together the enzyme’s different protein subunits
bringing together proteins that function in the same cell process
Chapter 16
cell communication
cells communicate with each other via signals (ligands)
the signal is sent by the signaling cell to be received by the target cell (receptors)
Cell signaling: the molecular mechanics by which cells detect and respond to external stimuli and send messages to other cells
Signal transduction: conversion of an impulse or stimulus from one physical/chemical form to another; process by which a cell responds to an extracellular signal
depends on receptor, intracellular relay system and targets
target cells have proteins called receptors that recognize and respond to signal molecules
different types of signals: endocrine, paracrine, autocrine, neuronal, contact-dependent
different types of ligands/receptors: small hydrophobic (intra-cellular receptors) and large hydrophilic (cell surface receptors)
extracellular signal molecules: any molecule outside of the cell that can elicit a response inside of the cell when the molecule binds to a receptor
can be proteins peptides, amino acids, nucleotides, steroids, fatty acids, and gasses
transported throughout the bloodstream through hormones
in animals, hormones are secreted into the bloodstream and distributed throughout the body
types of signals
paracrine signals are released by cells into the extracellular fluid
signaling cell releases short-lived ligands (e.g. growth factors) that affect nearby target cells (local)
secreted to extracellular matrix
involved in wound healing and regulating inflammation at site of infection
autocrine signaling: cells that respond to the local mediators that they themselves produce
cell sends signals to itself
not very common in physiological settings
T cells of immune system (macrophages) Interleukin-6 secretion
common in disease: cancer cells - angiogenesis
neuronal signaling: delivers message over long distances - transmitted electrically along a nerve cell axon. when this electrical signal reaches the nerve terminal, it causes the release of neurotransmitters onto adjacent target cells
delivers messages quickly and specifically to individual target cells through private lines
the axon of a neuron terminates at specialized junctions (synapses) on target cells
on reaching the axon terminal, these electrical signals are converted into a chemical form; each electrical impulse stimulates the nerve terminal to release a pulse of an extracellular signal molecule called neurotransmitter
neurotransmitter then diffuses across the narrow gap that separates the membrane of the axon terminal from that of the target cell, reaching its destination in less than 1 millisecond
contact-dependent signaling: most short ranged type of cell communication that makes direct physical contact through signal molecules in the plasma membrane of the signaling cell and receptor proteins embedded in the plasma membrane of the target cell
allows adjacent cells that are initially similar to become specialized to form different cell types
important early in development and cell differentiation
contact-dependent signaling in development:
controls nerve-cell production during embryonic development of fruit flies drosophila
in the fly embryo, a sheet of epithelial cells eventually specialize to become nerve cells
isolated cells begin to differentiate while neighboring cells remain unspecialized
lateral inhibition by contact-dependent signaling
notch signaling
takes a more direct route to control gene expression, studied in drosophila
acts as transcription regulator
pathway that regulates cell proliferation, cell fate, differentiation, and cell death in all metazoans
delta receptor (notch) itself is a transcription regulator
gets cleaved upon receiving signal and the C-terminal tail moves to the nucleus to regulate transcription
receptors
receptor: protein that recognizes and responds to a specific signal molecule
whether a cell responds to a signal molecule depends on whether it possesses a receptor
extracellular signal molecules can be divided into 2 major classes:
cell-surface(membrane) receptors - large/hydrophilic: rely on receptors on the surface of the target cell to relay their message across the plasma membrane
small/hydrophobic: pass through the plasma membrane and into the cytosol of the target cell, where they bind to intracellular receptor proteins
how a cell reacts to a particular signal depends on the set of intracellular signaling molecules each cell-surface receptor produces and how these molecules alter the activity of effector proteins, which have a direct effect on the behavior of the target cell
cells can respond to the same signal in two different ways
the information by the signal depends on how the target cell receives and interprets the signal
rapid responses from cells are possible because the signal affects the activity of proteins that are already present inside the target cell
majority of extracellular signal molecules are proteins, peptides, or small hydrophilic molecules that bind to cell-surface receptors that span the plasma membrane
transmembrane receptors detect a signal on the outside and relay a message across the membrane into the interior of the cell
the receptor protein performs the primary step in signal transduction; it recognizes an extracellular signal molecule and generates a different type of intracellular signal molecule
the resulting intracellular signaling sends the message downstream from one intracellular signaling molecule to another, each activating/generating the next signaling molecule in the pathway until a metabolic enzyme is kicked into action
the cytoskeleton is tweaked into a new configuration, or a gene is switched on or off
this final outcome is called the response of a cell
interpreting signals
cells are bombarded by signals constantly, but they only respond to specific signals in a certain manner
expression of specific receptors
intracellular relay system
intracellular targets vary
same signal - different effects on different cells
a typical cells contains thousands of copies of different receptors
this allows it to respond to combinations of signals, this is called “tailoring” of a cell’s response
when the signal response is cell growth or division - requires triggering gene expression
skeletal muscles contract within ms - signal affect the activity of proteins that are already there
signaling by steroid hormones
steroid hormones are secreted by three “steroid glands”- the adrenal cortex, testes, and ovaries
derived from cholesterol
bind to the intracellular receptors that belong to the nuclear superfamily of transcription regulators
cortisol is a hormone released by adrenal gland in starvation, stress, exercise, that signals to our liver to increase the production of glucose from amino acids - upregulation of gluconeogenesis genes
intracellular signaling pathways
most signals are hydrophilic (proteins, peptides) that recognize cell-surface TM receptors
signal relayed from one intracellular signaling molecule to another - signaling cascade
affect targets (effectors) and activate one or more intracellular pathways (same signal, different responses)
some of these signaling molecules interact with specific effector proteins, altering them to change behavior of cells in various ways
set of proteins and small-molecule second messengers that interact with each other to relay a signal from the cell membrane to its final destination
they can relay the signal onward and thereby help spread it through the cell. scaffold proteins can assist by bringing together the components needed to propagate the signal
they can amplify the signal received, making it stronger, so that a few extracellular signal molecules are enough to evoke a large intracellular response
they can detect signals from more than one intracellular signaling pathway and integrate them before relaying a signal onward
they can distribute the signal to more than one effector protein, creating branches in the information flow diagram and evoking a complex response
they can engage in feedback, modulating the response to the signal by regulating the activity of components upstream in the signaling pathway
feedback can occur anywhere in the signaling pathway and can either boost or weaken the response to the signal
in positive feedback, a component that lies downstream in the pathway acts on an earlier component in the same pathway to enhance the response to the initial signal
in negative feedback, a downstream component acts to inhibit an earlier component in the pathway to finish the response to the initial signal
positive feedback can generate all-or-none switch like responses whereas negative feedback can generate responses that oscillate on and off as the activities or concentrations of the inhibitory components rise and fall
Molecular switches
intracellular signaling protein that toggles between an active and inactive state in response to receiving a signal
for every activation step along the pathway, there exists an inactivation mechanism
proteins that act as molecular switches fall into one of two classes
proteins that are activated/inactivated by phosphorylation: the switch goes in one direction by a protein kinase, which covalently attaches a phosphate group onto the switch protein, and in the opposite direction by a protein phosphate, which takes the phosphate off again. the activity depends on the balance between the activities of the protein kinases that phosphorylate it and the protein phosphatase that dephosphorylate it
GTP-binding proteins: activity determined by its association with either GTP or GDP. includes both trimeric G proteins and monomeric GTPases such as Ras. G proteins relay messages from G-protein-coupled receptors. monomeric GTPases help relay their signals and are aided by regulatory proteins that help them bind and hydrolyze GTP: guanine nucleotide exchange factors (GEFs), which activate the switches by promoting the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) turn them off by promoting GTP hydrolysis
guanine nucleotide exchange factors (GEFs)
activate GTP-binding proteins by promoting exchange of GDP for GTP
GTPase-activating proteins (GAPs)
turn GTP-binding proteins off by promoting GTP hydrolysis
Cell-surface receptors
bind to extracellular signal and transduce the message via intercellular proteins to alter cell behavior or properties
Ion-channel coupled receptors
enzyme-coupled receptors
G-protein coupled receptors
Ion-chanel coupled receptors
alter membrane permeability to certain ions thus changing membrane potential
open to let specific ions such as K+, Na+, Ca2+, or Cl- to pass through the membrane in response to the binding of ligand (neurotransmitter)
acetylcholine receptor in the skeletal muscles convert chemical to electrical signal
enzyme-coupled receptors
activate enzymes in response to signal or themselves act as enzymes
usually one-pass transmembrane proteins
many of these enzymes are kinases (tyrosine kinases or serine/threonine kinases)
ex include the IRE1 and PERK in UPR
G-protein couple receptors
largest family of cell surface receptors
all G-protein coupled receptors (GPRs) have similar structures (7-pass transmembrane proteins)
embedded in the membrane by seven transmembrane a helices
function by activating membrane-bound trimeric G-proteins (or GTP-binding proteins) which then activates (or inhibits) an enzyme or ion channel initiating a signaling cascade
respond to numerous and diverse signals: hormones, neurotransmitters, local mediators
attractive targets for drug development
activate G-proteins: made of three subunits two of which are tethered to PM by lipidation: a, b, and g
GCPR
exist in two forms
active (when GTP is bound to a subunit) and inactive (when GDP is bound to a subunit)
Turning off GPCRs:
binding to GTP activates a-subunit: a-subunit dissociates from by complex
both interacts with membrane proteins that can further relay a signal: Gs stimulate and Gi inhibit targets
the longer target proteins are bound to a-subunit or to by complex, the more prolonged the signal will be
to turn off the pathway, a-subunit (intrinsic GTPase) hydrolyzes its bound GTP to GDP and Pi
constitutively active G protein signaling cannot be turned off
GPCR activates the signaling pathway by reducing affinity to GDP and increasing affinity to GTP
pathway is turned off by intrinsic a-subunit GTPase activity which hydrolyzes GTP to GDP
turning on and off a signaling pathway are equally important
the targets of activated Gs and Gi can be either ion channels or enzymes
channel coupled GPCR signaling is immediate and fast
most common enzyme targets are adenylyl cyclase and PLC phospholipase C
ex. coupling GCPR to K+ channels
acetylcholine (released by nerve cells) binds to its GCPR on heart muscle cells - activation of G protein
this activates bg complex which binds to K+ channels and holds them in open confirmations
permeability to K+ increased and K+ is fluxed outside the cell
hyperpolarization causes action potential to slow down - contraction slows down
ex. coupling GCPRs to adenylyl cyclase
converts ATP to cAMP (second messenger)
second messengers: intracellular signaling molecules released by the cell in response to extracellular signaling molecules - the first messengers
diffuse inside cell and amplify the signal
cAMP phosphodiesterase breaks down cAMP to terminate the signal
GTP-bound a subunit activates adenylyl cyclase to produce cAMP
cAMP binds to and activates the enzyme protein kinase A (PKA)
PKA phosphorylated downstream targets (proteins)
phosphorylation changes protein activity
e.g. epinephrin stimulates glycogen breakdown in skeletal muscles (glycogenesis)
ex. G-proteins coupling to phospholipase C
phospholipase C: enzyme associated with the plasma membrane that generates two small messenger molecules in response to activation
cleaves inositol phospholipids to inositol triphosphate (IP3) and diacylglycerol (DAG)
bound to cytosolic face of plasma membrane
IP3: small intracellular signaling molecule that triggers the release of Ca2+ from the ER into cytosol; produced when a signal molecule activates a membrane-bound protein - phospholipase C
water-soluble sugar phosphate that is released into the cytosol; there it binds to and opens Ca2+ channels that are embedded in the ER membrane
DAG: small messenger molecule produced by the cleavage of membrane inositol phospholipids in response to extracellular signals; helps activate protein kinase C
lipid that remains embedded in the plasma membrane after it is produced by phospholipase C; there, it helps recuit and activate a protein kinase, which translocates from the cytosol to plasma membrane
IP3 binds to Ca2+ channels at the ER and promotes the release of Ca2+ into the cytosol
Ca2+ is an important signaling molecule (second messenger)
GPCRs that activate phospholipase C exert their effects through G protein called Gq
protein kinase C: enzyme that phosphorylates target proteins in response to a rise in diacylglycerol and Ca2+ ions
GPCR signaling
type of GPCR signaling generate nitric oxide (NO)
NO: locally acting gaseous signal molecule that diffuses across cell membranes to affect the activity of intracellular proteins
distance of NO is limited by its reaction with water and oxygen, which converts NO into nitrates and nitrites
acetylcholine binds to GPCR on surface of epithelial cells
stimulates increase in cytosolic Ca2+
activates NO synthase to make NO from Arg
NO diffused to neighboring cardiac muscle cells
bind to guanylyl cyclase which converts GTP to cGMP
causes smooth muscle cells to relax; vasodilation, lower blood pressure
Calmodulin: relaying the Ca2+ signal
calmodulin: small Ca2+ binding protein that modifies the activity of many target proteins in response to changes in Ca2+ concentration
when Ca2+ binds to calmodulin, the protein undergoes conformational changes and alters the activity of other proteins
Ca2+ is a second messenger that often relays its signals using calmodulin
4 binding sites for Ca2+
activates Ca2+/calmodulin-dependent protein kinases (CaM-kinases)
CaM Kinases: enzyme that phosphorylates target proteins in response to an increase in Ca2+ ion concentration through its interaction with the Ca2+ binding protein calmodulin
activated by binding to calmodulin complexed with Ca2+, they then influence other processes in the cell by phosphorylating selected proteins
play important role in memory
cAMP
functions by inducing PKA-dependent protein phosphorylation
responses involve changes in gene expression that take minutes or hours to develop
in these slow responses PKA typically phosphorylates transcription regulators, proteins that activate the transcription selected genes
depending on what proteins get phosphorylated the output signal will vary
cAMP can activate transcription by regulating activity of transcription factors
mediates different cellular processes
adrenaline - G protein-couple receptor - G protein
adrenaline (epinephrine) hormone secreted by adrenal gland
affects different cells (adrenergic receptors) differently because of different response/effector proteins
the response helps prepare the body for sudden action
mediates different cellular responses because different cell types have different targets
Enzyme-couple receptors
receptor tyrosine kinases (RTKs): enzyme coupled receptor in which the intracellular domain has a tyrosine kinase activity, which is activated by ligand binding to the receptor’s extracellular domain
most common
binding of signal molecule to receptor causes them to dimerize 9to associate together)
dimerization activates the kinase domain on the cytosolic side - receptors phosphorylate each other one specific tyrosine
P-Tyr serves a docking site for target protein (SH2 domain)
Tyr phosphatase turns OFF the signal
in some cases, signal is turned off by recycling the whole receptor
enzyme-coupled receptor has to switch to the enzyme activity of its intracellular domain
have 1 transmembrane segment unlike GPCRs
binding of an extracellular signal molecule causes two receptor molecules to come together in the plasma membrane, forming a dimer; this also causes the two intracellular tails of the receptors to come together and activate their kinase domains
each tail phosphorylates the other; occurring on specific tyrosines
the phosphorylation triggers an assembly of intracellular signaling complex on the cytosolic tails of the receptors, which serves as a docking site for signaling proteins
either becomes scaffolds or activated on binding to the receptors to propagate the signal
socked intracellular signaling proteins have a specialized interaction domain, which recognized specific phosphorylated tyrosines on the receptor tails
allows intracellular signaling proteins to recognize phosphorylated lipids that are produced on the cytosolic side of plasma membrane
RTK
Ras activation is an example of RTK signaling (ex 1)
Ras: one of a large family of small GTP-binding proteins that helps relay signals from cell surface receptors to the nucleus
attached by a lipid tail to the cytosolic face of plasma membrane
part of family of GTP-binding proteins called monomeric GTPases
activation requires recruitment of Ras-GEF to activated RTK
all RTKs activate Ras, including platelet-derived growth factor (PDGF) receptors, and nerve growth factor (NGF) receptors
monomeric GTPases: small GTP-binding protein; proteins of this family such as Ras and Rho are part of many different signaling pathways
Ras cycles between 2 distinct conformational states; active when GTP is bound and inactive when GDP is bound
interactions with activating protein Ras GEF encourages Ras to exchange its GDP for GTP, switching Ras to its activated state
after a delay, Ras is switch off by a GAP called Ras GAO, which promotes the hydrolysis of its bound GTP to GDP
in its active state, Ras initiates a phosphorylation cascade in which a series of serine/threonine kinases phosphorylate and activate one another in sequence
Ras amplification cascade
Ras activates MAP kinase MAPK signaling
in its active state, Ras promotes activation of phosphorylation cascade, in which series of serine/threonine protein kinases phosphorylate and activate one another
Ras is an oncogene
receptor serine/threonine kinases: enzyme-coupled receptor that phosphorylates target proteins on serine or threonine
MAP-kinase signaling module: set of 3 interlinked protein kinases that allows cells to respond to extracellular signal molecules that stimulate proliferation; includes a mitogen-activated protein kinase (MAP kinase), a MAP kinase kinase, and a MAP kinase kinase kinase
MAP-kinase: mitogen-activated protein kinase; signaling molecule that is the final kinase in a three-kinase sequence called the MAP-kinase signaling module
RTKs can activate the PI-3-Kinase-Akt pathway (ex 2)
PI 3-kinase (phosphoinositide 3-kinase) phosphorylates inositol phospholipid, which recruits the serine/threonine protein kinase Akt (protein kinase B) that is activated by protein kinases 1 and 2
Phosphoinositide 3 kinase (PI3-kinase): enzyme that phosphorylates inositol phospholipids in the plasma membrane, generating docking sites for intracellular signaling proteins that promote cell growth and survival
serve as docking sits for specific intracellular proteins which relocate from the cytosol to the plasma membrane
once activated, AKT is released from the plasma membrane to perform its function in the cytosol
Akt directly phosphorylates protein called Bad and inactivates signaling pathway
unphosphorylated Bad promotes cell death by binding and inhibiting Bcl2 which otherwise suppresses apoptosis (form of cell death)
phosphorylation and inactivation of Bad thus promoting survival in response to extracellular signals
Akt/PKB: promotes the growth and survival by inactivating the signaling proteins it phosphorylates
phosphorylation by Akt promotes cell survival by inactivating a protein that promotes cell death (such in protein Bad)
PI-3-kinase-Akt signaling pathway stimulates cells to grow in size; it does so by indirectly activating a large serine/threonine kinase called Tor
Tor stimulates cells to grow both by enhancing protein synthesis and by inhibiting protein degradation
Akt indirectly activates TOR by phosphorylating and inhibiting protein that helps to keep Tor shut down
binding of growth factors to RTK
when TOR is active → cell growth
when TOR is inactive → autophagy
Chapter 18
Cell Division
cells coordinate growth and division
duplication of DNA and segregating into two genetically identical daughter cells
cells grow in size before they divide
duplication of other components, synthesize of new proteins and organelles
time it takes for a cell to divide depends on the cell type
early fly embryo cells: 8 minutes
early frog embryo cells: 30 minutes
mammalian intestinal epithelial cells: 12 hours
mammalian fibroblasts in culture: 20 hours
phases of cell cycle
Interphase: long period of the cell cycle between one mitosis (M) and the next
includes G1 phase, S phase, and G2 phase
cells duplicate their content and grow in size, making proteins and organelles
during interphase, cell continues to transcribe genes, synthesize proteins, and grow mass
M phase: period of eukaryotic cell cycle during which the nucleus and cytoplasm divide to produce two daughter cells (mitosis/cytokinesis)
includes mitosis (division of nucleus) and cytokinesis (division of cell)
S (synthesis) phase: period during a eukaryotic cell cycle in which DNA is synthesized; replicates DNA
G1 phase: falls between the end to cytokinesis and start of DNA synthesis
G2 phase: falls between the end of DNA synthesis and the beginning of mitosis
during G1 and G2, cells monitor conditions and assess whether it is suitable for growth
G0 phase: a cell that enters the G0 phase enters a state of cell cycle quiescence
cells are not dividing or preparing to divide
checkpoints
eukaryotic cells possess cell-cycle control system that can stop the cycle at various checkpoints
cell-cycle control system: makes sure that the cell cycle occurs in the correct sequence using critical points of the cycle by feedback from the process being performed
one cycle ends before the next one begins
DNA replication must finish before nucleus can divide
If DNA synthesis is slowed, mitosis and cell division must also be slowed
if DNA is damaged, the cycle must slow until repair is complete
cycle put on hold in G1, S, or G2 so the cell can repair the damage either before DNA replication is started/completed, or before the cell enters M phase
in the late G1 phase, the control system confirms that the environment is favorable for proliferation before proceeding to replicate its DNA
the point where cells commit to begin and complete a full cell cycle
start - important transition at the end of the G1 phase of cell cycle; passage through this transition commits the cell to enter the cell cycle and continue to S phase
at the second main transition from G2 to M phase, the control system confirms that the DNA is undamaged and fully replicated
at the third transition point, midway through mitosis, the cell-cycle control machinery confirms that the duplicated chromosomes are properly attached to the mitotic spindle, before the spindle pulls the chromosomes apart and segregates them into the two daughter cells
cyclins/cdks
cyclins: regulatory protein whose concentration rises and falls at specific times during the eukaryotic cell cycle
help control progression from one stage of cell cycle to the next by binding to cyclin-dependent protein kinases (cdks)
cyclin-dependent protein kinases (Cdks): enzyme that, when complexed with a regulatory cyclin protein, can trigger various events in the cell division cycle by phosphorylating proteins
regulated by phosphorylation and dephosphorylation
activities depend on regulatory proteins called cyclin
must bind to cyclin to become enzymatically active
cyclins also direct Cdks to target proteins
cyclins concentration very during cell cycle
M cyclin: acts in G2 to trigger entry into M phase; regulatory protein that binds to mitotic Cdk to form M-Cdk, the protein complex that triggers the M phase of cell cycle
M-cyclin + Cdk
G1/S cyclin: regulatory protein that helps to launch the S phase of the cell; bind to Cdk protein to form G1/S-Cdk and S-Cdk
G1/S-cyclin + Cdk
G1 cyclin: regulatory protein that helps drive a cell through the first gap phase of the cell cycle towards the S phase
G1-cyclin + Cdk
S cyclin and G1/S cyclin Cdks help launch S phase
S cyclin rise in late G1 and remain high through S, G2, and early mitosis
M cyclin rises as the cell begins to enter mitosis
activity of cyclin
Cyclins concentrations rise and fall in a cyclic manner during cell cycle
regulated by transcription and proteolysis (degradation)
concentrations regulate Cdks activities which then phosphorylate and activate different Cdk targets
concentrations of Cdks don’t change, but their activities are dependent on cyclin concentration changes
concentration of a given protein in the cell is determined by the rate at which the protein is synthesized and the rate at which it is degraded
anaphase-promoting complex, or cyclosome: a protein complex that triggers the separation of sister chromatids and orchestrates the timed destruction of proteins that control progress through the cell cycle
the complex catalyzes the ubiquitylation of its targets
complex tags the cyclins with a chain of ubiquitin, which are directed to proteasomes, where they are rapidly degraded
ubiquitylation and degradation of a cyclin returns its Cdk to inactive state
cyclin destruction can also help drive the transition from one phase cell to another
M-cyclin degradation and the resulting inactivation of M-Cdk leads the cell out of mitosis
activity of associated cyclin-Cdk complexes tend to switch on abruptly at the appropriate time in cell cycle
cyclin-Cdk complex contains inhibitory phosphates, and to become active, the Cdk must be dephosphorylated by a specific protein phosphatase
cyclin-Cdk complex is not phosphorylated and is inactive when first formed
protein kinases phosphorylate Cdk keeping the complex in an inactive state
cell cycle inhibitors can be used to pause or delay the transition through cell cycle stages if the conditions are not favorable for cell division
p27 binds to an activated complex and it inhibits its activity
this complex is no longer able to phosphorylate downstream target proteins required for progress of cell cycle
G1
cells metabolically active, cell growth and damage repair are taking place
based on signals, cells decide whether to continue to S-phase or stay temporarily in G0 or G1
G0 is a prolonged non-proliferative state
from here cells can re-enter the cell cycle (e.g. liver cells)
some cells never leave G0
e.g. mature neurons and muscle cells
once past G1-S transition, cells commit to division
mitogens: extracellular signals that stimulate cells to multiply
if deprived of signal, the cell cycle arrests in G1
if deprive of mitogens for long enough, it will withdraw from the cell cycle and enter G0
by the end of M phase, cells inactivate all Cdks ensuring cells cannot commit to another round of cell division before going to G1. mitogens induce cells to replicate and divide by switching ON signaling pathways that stimulate the synthesis of G1 cyclins and G1/S cyclins and other proteins involved in DNA replication
if DNA is damaged, the cell cycle control system uses several mechanisms to halt progression into S phase and cell proliferation until damage is repaired
S phase
to begin replicating DNA, the cell needs the origin recognition complex (ORC), which stays bound to origins of replication throughout the cell cycle
origin recognition complex (ORC): remains perched on the replication origins throughout the cell cycle, to prepare for DNA replication, the ORC recruits a protein called Cdc6, whose concentration rises early in G1
in early G1, Cdc6 binds to the ORC, and together these proteins load additional proteins to form prereplicative complex
these proteins position the DNA helicases that will open up the double helix at the origin of replication
the signal to commence replication comes from S-Cdk, which triggers S phase
S-Cdk is assembled and activated at the end of G1
at the start of S phase, S-Cdk triggers the initiation of DNA replication by guiding the assembly of DNA polymerase and other proteins that initiate DNA synthesis at replication fork
also prevents the replication from occurring again at the same origin by phosphorylating Cdc6
M phase
M-Cdk starts accumulating in G2
along with binding to M-cyclin, mitotic Cdk (M-Cdk) also needs to be dephosphorylated for activation
M-Cdk is activated in M phase
once activated, M-Cdk phosphorylates and activates phosphatase (Cdc 25)
M-Cdk also activates APC/C complex to turn itself off by degrading M-cyclin at end of M phase
activation of M-Cdk is inhibited by phosphorylation at particular sites
for cell to progress into mitosis, these inhibitory phosphates must be removed by an activating protein phosphatase called Cdc25
if DNA replication stalls, the presence of single stranded DNA at replication fork triggers a DNA damage response
part of this response includes inhibition of the phosphatase Cdc25, which prevents the removal of the inhibitory phosphates from M-Cdk
as a result, M-Cdk remains inactive and M phase is delayed until DNA replication is complete and any DNA damage is repaired
once a cell has successfully replicated its DNA in S phase and progressed through G2, it is ready to enter M phase
if DNA replication is not done properly → Cdc25 remains inactive and cannot dephosphorylate M-Cdk
Cdc25 activates M-Cdks by removing the inhibitory phosphates from them
cohesions and condensins
DNA replication takes place in S phase
following replication, each chromosome consists of two identical sister chromatids
held together by protein complexes called cohesion
sister chromatids: copy of chromosome produced by DNA replication that still remains bound to the other copy
contain a double-stranded molecule of DNA along with its associated proteins
held together by cohesions
cohesions: ring shaped SMC protein complex that organizes interphase chromosomes into a long series of large chromatin loops; in addition, a special subset of cohesions hold together the sister chromatids after the DNA has been replicated
defects in cohesions lead to chromosome segregation errors - aneuploidy
encircle the two sister chromatids, tying them together
condensins: ring shaped SMC protein complex that compacts duplicated chromosomes for segregation by forming both loops within loops
carry out chromosome condensation early in mitosis
assemble along each individual sister chromatid, helping each of these double helices to coil up into a more compact form
condensation is accompanied by the partial removal of the cohesion complexes from along each chromosome’s arms
this resolution allows the sisters to remain tightly associated while they are attached to the spindle but then move apart safely in late mitosis
after the duplicated chromosomes condensed, the mitotic spindle carries out nuclear division (mitosis), and in animals, the contractile ring divides the entire cell into 2 (cytokinesis)
mitotic spindle is composed of microtubules and the various proteins that interact with them, including microtubule-associated motor proteins
contractile ring consists mainly of actin and myosin filaments arranged ina. ring around the equator of cell
starts to assemble just beneath the plasma membrane towards the end of mitosis
as ring contracts, it pulls the membrane inward, splitting the cell into two
M phase stages
consists of 6 stages
prophase
prometaphase
metaphase
anaphase
telophase
cytokinesis → begins before mitosis ends
centrosome: microtubule-organizing center that sits near the nucleus in an animal cell; during the cell cycle, this structures duplicates to form the two poles of the mitotic spindle
centrosome duplication begins at the same time as DNA replication, and the process is triggered by the same Cdks (G1/S-Cdk and S-Cdk) that initiate DNA replication
remain together as a single complex until M-phase begins
separate to opposite poles of the dividing cell and make the asters
aster: star shaped array or microtubules from a centrosome or from a pole of mitotic spindle
assembly of mitotic spindle depends on the microtubules
two cytoskeletal structures must form to carry out mitosis and cytokinesis that occur in M phase
mitotic spindle directs movement and separation of sister chromatids during mitosis; composed of microtubules
contractile ring contracts to divide cytoplasmic contents during cytokinesis; composed of actin and myosin filaments
prophase: first stage of mitosis during which the duplicated chromosomes condense and the mitotic spindle forms
outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have begun to move apart
prometaphase: stage of mitosis in which the nuclear envelope breaks down and duplicated chromosomes are captured by the spindle microtubules; precedes metaphase
spindle microtubules attach to the chromosome at their kinetochores
kinetochore: protein complex that assembles during late prophase on the centromere of a condensed mitotic chromosome (+ end); the site to which spindle microtubules attach
recognizes the special DNA sequence that forms a centromere
each duplicated chromosome has two kinetochores → one on each sister chromatid
the attachment to opposite poles, called bi-orientation, generates tension on the kinetochores, which are being pulled in opposite directions
this tension signals to the sister kinetochores that they are attached correctly and are ready to be separated
during prometaphase, the duplicated chromosomes now attached to the mitotic spindle align at the equator of the spindle
forms the metaphase plate
the minus end of microtubules are anchored in the centrosome, while the free plus ends are dynamically unstable
kinetochore microtubules bind to the centromeres
non-kinetochore microtubules (interpolar)
motor proteins and other microtubule-associated proteins cross-link the interpolar microtubules and stabilize the plus ends by decreasing their depolymerization
astral microtubules attach to the cell cortex
structure of mitotic spindle
aster microtubules: short, radiate from centrosomes, interact with cell cortex
interpolar microtubules: interact with each other, from opposite poles of spindle
motor proteins and other microtubule-associated proteins stabilize interpolar microtubules
kinetochore microtubules
metaphase: stage of mitosis in which chromosomes are properly attached to the mitotic spindle at the equator but have not yet segregated towards opposite poles
chromosomes are suspended in the middle under tension
chromosomes aligned at equator → metaphase plate
anaphase: stage of mitosis during which the two sets of chromosomes separate and are pulled towards opposite ends of the dividing cell
breaks cohesion link between sister chromatids
cohesion linkage destroyed by a protease called separase
this protease is held in an active state by an inhibitory protein called securin
at the beginning of anaphase, securin is targeted for destruction by anaphase promoting complex (APC) - the same protein complex that marks M-cyclin for degradation
APC indirectly promotes cohesion breakdown
APC catalyzes ubiquitylation and destruction of inhibitory protein, securin
once securin has been destroyed, separase is then free to sever the cohesion linkages
anaphase A: kinetochore microtubules shorten and the attached chromosomes move poleward; driving force provided by loss of tubulin subunits from both ends of the kinetochore
anaphase B: spindle poles move apart, further segregating the 2 sets of chromosomes; driving forces provided by two sets of motor proteins (members of the kinesin and dynein families) operating on different types of spindle microtubules
kinesin proteins slide microtubules from opposite poles past one another at the equator
dynein proteins move along astral microtubules to pull the poles apart
telophase: final stage of mitosis in which the two sets of separated chromosomes decondense and become enclosed by a nuclear envelope
the nuclear pore proteins and nuclear lamins that are phosphorylated during prometaphase are now dephosphorylated, which allows them to reassemble and rebuild the nuclear envelope and lamina
once nuclear envelope reestablished, the pores restore the localization of cytosolic and nuclear proteins and the condensed chromosomes decondense into their interphase state
cytokinesis: process by which the cytoplasm divides in 2 to form individual daughter cells
depends on transient structure based on actin and myosin filaments, also known as contractile ring, which pinches the cell into two daughter cells, each with one nucleus
completes M phase, begins in anaphase
first visible sign of cytokinesis is a furrowing of the plasma membrane (anaphase)
ensures that the cleavage furrow cuts between the segregated chromosomes to give each daughter cell identical and complete set of chromosomes
plane of cleavage and timing of cytokinesis is determined by mitotic spindle
in plant cells: new cell wall forms at the equator to divide the cell
separated through a new wall that forms inside of dividing cell, compared to animal cells that are separated through contractile ring
planes of cell division and cell enlargement determine the final form of the plant
process guided by phragmoplast: in a dividing cell, structure containing microtubules and membrane vesicles that guides the formation of a new cell wall
apoptosis and necrosis
three fundamental processes largely determine organ and body size
cell growth
cell division
cell death
apoptosis: tightly controlled form of programmed cell death that allows cells to be eliminated from an adult or developing organism
can remove cells that aren’t needed
happens for many reasons
cells in developing mouse paw sculpt the digits
metamorphosis; tadpole to frog, cells in tail die
cells lining the intestine are constantly recycling
adjust the number of developing nerve cells to target cells
cells in the midst of apoptosis may develop blebs on its surface, but then shrinks and condenses
necrosis: cells that due as a result of acute injury; typically swell and burst, spewing their contents across neighboring cells
uncontrolled cell death
apoptosis signaling cascade
apoptosis in cell involved a signaling cascade, which activates different caspases (suicide proteases) that induce apoptosis
caspases: one of a family of proteases that, when activated mediates the desctriction of the cell by apoptosis
inactive and active in response to signals that induce apoptosis
activated caspase can activate more procaspaces, kicking off amplifying, proteolytic cascade
some activated caspases break down other key proteins in the cell, leading to the controlled death of a cell
two types of caspases work together to take a cell apart
initiator caspases cleave
executioner caspases; one form of this target the lamin proteins that form the nuclear lamina underlying the nuclear envelope, which causes the irreversible breakdown of the nuclear lamina and allows nucleases to enter the nucleus and break down the DNA
Bcl2 inhibits caspases and apoptosis
Bcl2: related group of intracellular proteins that regulates apoptosis; main proteins that regulate the activation of caspases
some promote caspase activation where others inhibit the process
Bax and Bak are death promoting members of the Bcl2 family that activate caspases and apoptosis
induce release of electron-transport protein “cytochrome c” from the mitochondria into the cytosol
other members of the Bcl2 family inhibit apoptosis by preventing Bax and Bak from releasing cytochrome c
cytochrome c molecules then activate initiator caspases, inducing cell death
extracellular signals can also induce apoptosis
active cell-surface receptor proteins known as death receptors (e.g. Fas)
Fas: present on the surface of many mammalian cells
activated by a membrane-bound protein called Fas ligand, present on the surface of specialized immune cells called killer lymphocytes
help regulate immune responses by inducing apoptosis in other immune cells
survival factors suppress apoptosis
act by binding to cell-surface receptors
the activated receptor activates transcription regulate in the cytosol
transcription regulator moves to the nucleus where it activates the gene encoding Bcl2