BIOL 4004 UMN Exam 2

A neuron cell and a liver cell share the same _____, but they have different _____

share the same genome, have different morphology

function by presumably expressing different sets of RNAs and proteins

Transcription regulators account for about ______ of all protein-coding genes.

10%

How do transcription regulators work

the transcription regulators recognize DNA cis-regulatory sequences and make their contacts with the major grooves.

The protein also contacts the minor groove and phosphates in the backbone

Dimerization of Transcription Regulators Increases Their

affinity and specificity for DNA

dimer (transcription regulators) finds Nanog cis-regulatory sequence in genome, specific for dimer types

A Eukaryotic Gene Control Region Consists of a ______ Plus Many ________ sequences, and functions to regulate..

Consists of a Promoter Plus Many cis-Regulatory Sequences

has co-activators, DNA looping, and transcription regulators
All of these ultimately regulate the recruitment of RNA polymerase II by the general transcription factors to the promoter

co-activators

proteins that do not bind DNA themselves but assemble on other DNA binding transcription regulators

Gene Expression regulated at steps in the pathway from DNA to RNA to protein

1: transcriptional control

2: RNA-processing control

4: translational control

5: mRNA degradation control

dont worry about others for now

Eukaryotic Transcription Repressors Can Inhibit Transcription in Several Ways (6)

1: competitive DNA binding
(repressor binding site overlapping w/ activator binding site)

2: masking the activation surface
(repressor binding site too close to activator binding site, represses)

3: direct interaction with the general transcription factors
(repressor binding site in place where repressor directly interacts w/ TFIID)

4: recruitment of chromatin remodeling complexes
(remodels chromatin into heterochromatin)

5: recruitment of histone deacetylases- histone deacetylation

6: recruitment of histone methyl transferase - histone methylation, protein bind to methylated histones


3-6: Chromatin remodeling and histone de-acetylation or methylation decrease the accessibility of TATA box to the general transcription factors and RNA polymerases

Insulator DNA Sequences Prevent Eukaryotic Transcription Regulators from Influencing _____ Genes

Insulator DNA Sequences Prevent Eukaryotic Transcription Regulators from Influencing Distant Genes

barrier sequences

Barrier sequences prevent the spread of heterochromatin The tethering to a fixed site stops the spread of heterochromatin.

By recruiting histone-modifying enzymes, barriers erase the histone marks that are required for heterochromatin to spread

Complex Genetic Switches That Regulate Drosophila Development Are Built Up from _________

Complex Genetic Switches That Regulate Drosophila Development Are Built Up from Smaller Molecules

The Drosophila Even-skipped gene (eve) is expressed in seven precisely positioned stripes as shown in green. Giant gene is expressed in the head and tail as shown in red, whereas yellow indicates the presence of both.

A 20 kb region upstream of the eve gene contains multiple cis-regulatory sequences, each specifies its specific expression in one stripe

The Drosophila Eve Gene Is Regulated by __________

Combinatorial Controls

Combinatorial Gene Control Creates Many Different Cell Types

Combinatorial Gene Control Creates ________

many different cell types

Liver cells (A) were converted into neuronal cells (B) via the artificial expression of three nerve-specific transcription factors that activate many nerve-specific genes and repress many liver-specific genes.

In drosophila, the expression of the Eyeless gene in precursor cells of the leg triggers the development of ________

an eye on the leg

Specialized Cell Types Can Be Experimentally Reprogrammed to Become:

Pluripotent Stem Cells

Stem cells: undifferentiated cells that can perpetuate themselves and give rise to other daughter cells capable of differentiating into specialized cells

a differentiated fibroblast cell can be induced to a ____

induced pluripotent stem cells iPS

1. genes encoding three transcription regulators introduced to and expressed in fibroblast nucleus

2. cells induced by further transcription regulators to differentiate in culture

fibroblast —> iPS cell —> muscle cell, neuron, fat cell, etc.

_____ leads to the depletion of a stem-cell population in the hair follicles of mice nervous system

stress

This discovery sheds light on why stress turns hair grey

Patterns of DNA Methylation Can Be ______ When Vertebrate Cells ______

Patterns of DNA Methylation Can Be Inherited When Vertebrate Cells Divide

DNA methylation steps

1. transcription regulator brings in a writer for histone modification, which is then read by code reader

2. the reader relays the writer to perform similar modification

3. at some moments the reader also brings in a de novo (new) DNA methylase

4. The methylated cytosine lies in the major groove and interferes with the binding of proteins

5. The binding of additional proteins to the methylated DNA adds further insulation

Genomic Imprinting Is Based on ________

DNA Methylation

Genomic imprinting: when the maternally inherited gene is active, its paternally inherited gene copy is silent or vice versa

The silence is due to DNA methlyation

If mutated only one gene copy matters

genomic imprinting steps

1. both parents express the same allele of gene A (from chromosome inherited from father)

2. removal of imprinting in germ cells, followed by meiosis

3. Female/male imprints established

4. Grow into mature individuals

5. Offspring differ in the allele of gene A that is expressed

basically when maternally inherited gene is active, paternally inherited gene copy is silent or vice versa

genomic imprinting silencing vs expression

methylation of insulator element leads to no CTCF binding, communication between cis-regulatory sequence and lgf2 gene established, gene expressed

if not methylated, CTCF binds insulator and blocks communication

synthesis of lncRNA can be blocked by methylation on Kcnq1 gene, leads to gene expression

if no methylatio on Kcnq1 gene lncRNA synthesized, alters chromatin, Kcnq1 silenced

Introduction and Alternative RNA Splicing Can Produce Different Forms of a Protein from the ____

same gene

alternative RNA splicing occurs for about 90% genes in human and may be critically regulated

A Change in the Site of RNA Transcript Cleavage and Poly-A Addition Can Change the ________ of a Protein

C-terminus

When the level of Cleavage stimulation factor (CstF) is low, it skips the first weak polyadenylation signal to produce a longer transcript.

A longer string of hydrophobic amino acids is retained as the membrane-bound domain.

When activated to produce antibodies, CstF level increases to cleavage the weak site

microRNAs (miRNAs) Regulate ____ and ____

Translation and Stability

There are about ____ 23-nt miRNAs in humans

1000

RNA Interference Can Direct ________ Formation

Heterochromatin formation

Many transposable elements and viruses produce double-strand RNA in their life cycles, triggering RNAi to against the invaders in plants, worms,and insects

steps in heterochromatin formation by RNA interference

protein complex (dicer) cleaves double stranded RNAs to siRNAs

siRNAs trigger the inhibition of translation and destruction of mRNAs as other miRNAs do

siRNAs also cause transcriptional silence. The short siRNAs interact with a group of proteins including argonaute to form RNA induced transcriptional silence (RITS) complex

The RITS complex directs the formation of heterochromatin

Long Noncoding RNAs Functions in the Cell

lncRNA does not code for proteins

lncRNAs carry proteins to specific RNA or DNA sequences through complementary base-pairing

Some lncRNAs regulate transcription in cis

Other lncRNAs diffuse from their sites of synthesis and act in trans

major lipids in cell membranes

Phosphoglycerides, Sphingolipids, and Sterols

Phosphoglycerides or phospholipids are derived from ______, whereas sphingomyelin is derived from _______ as _____

Phosphoglycerides or phospholipids are derived from glycerol

sphingomyelin is derived from sphingosine as a sphingolipid

__________ is the most abundant phospholipid

phosphatidylcholine

______ has a net negative charge

phosphotidylserine

cholesterol formula, model, and schematic drawing

C27H46O

plant membranes do not contain cholesterols and contain more __________

unsaturated fatty acids

The Lipid Bilayer Is a _________ fluid

two-dimensional

fatty acid tails, lipid head groups on either side

water molecules stay on both sides of the membrane

_______ channels allow the bulk flow of water molecules across the lipid bilayer

aquaporin channels

raft domain lipid bilayer

specialized domains or membrane regions involving protein-protein, protein-lipid and lipid-lipid interactions.

Raft domains have an increased membrane thickness.

GPI: glycosylphosphatidylinositol

lipid bilayer asymmetry

Outer layer: phosphatidylcholine, sphingomyelin

Inner layer: ethanolamine (terminal amino group), serine

The cytosolic (inner) side carries negative charges

Some lipid kinases phosphorylate its head group to form a binding site that recruits other proteins to the cell surface or cytosolic face

Membrane Proteins Can Be Associated with the Lipid Bilayer in Various Ways

Transmembrane proteins covalently attached to a fatty acid chain in the cytosolic side, multiple alpha-helices or a rolled-up beta-sheet

Protein can be anchored to the cytosolic surface by an amphilic alpha-helix

Protein can be attached to the bilayer by a lipid chain

Protein can associate with the membrane via GPI-anchor (glycosylphosphatidylinositol)

Peripheral proteins associate with other membrane proteins

how to determine membrane protein topology

make cell not permeable, treat with trypsin, it digests extracellular parts of proteins, do SDS-PAGE, find which proteins had most taken off them = probably extracellular facing

Lipid Anchors Control the _______ of Some Signaling Proteins

membrane localization

Myristoyl anchor lipid anchor

Myristic acid is 14-C unsaturated fatty acid and recruits Src family tyrosine kinase in the cytosolic face

Palmitoyl anchor lipid anchor

Palmitic acid is 16-C unsaturated fatty acid as the second anchor to recruit Src, and Src returns to cytosol when the signal is off

Farnesyl anchor lipid anchor

Farnesyl is a tri-prenyl group as a 15-carbon unsaturated hydrocarbon chain

in Most Transmembrane Proteins, the Polypeptide Chain Crosses the Lipid Bilayer in an ________ conformation

alpha-helical

Hydrophobic Amino acids with non-polar side chain align with hydrophobic core

Intra-molecular hydrogen bonds between adjacent peptide bonds stabilize the alpha-helical structure

Some β-Strands Form Large _________

channels

Multipass beta-strands form antiparallel beta-barrels normally in the outer membrane of bacteria, mitochondria and chloroplasts.

OmpA is a receptor for a bacterial virus

OMPLA is a lipase

Porin and FepA are transporters

many Membrane Proteins Are _______-ated

purpose of this modification?

glycosylated

Purpose primarily to help during protein folding, attachment onto membrane

Membrane Proteins Can Be Solubilized and Purified in _______

detergents

Three commonly used detergents are

sodium dodecyl sulfate (SDS), an anionic detergent (negatively charged), and Triton X-100 and β-octylglucoside, two nonionic detergents

______ detergents are used for solubilizing membrane proteins such as Na+/ K+ pump

steps for solubilizing Na+/K+ pump

mild non-ionic detergents

1. add detergent, membrane protein solubilized

2. add lipid-detergent to purify

3. removal of detergent

4. addition of phospholipids (mixed with detergent)

5. functional pump incorporated into phospholipid vesicle

Many Membrane Proteins _____ throughout the Plane of the Membrane

diffuse

Many membrane proteins rotate and move laterally. When mouse cells are fused with human cells, the two sets of proteins diffuse and mix in about half an hour

Cells Can Confine Proteins and Lipids to Specific _____ Within a _________

can confine proteins and lipids to specific domains within a membrane

In epithelial cells that line the gut or the tubules of the kidney, proteins are confined to the apical or lateral and basal surfaces due to tight junction

Four ways of restricting the lateral mobility of specific plasma membrane proteins

(A) The proteins can self-assemble into large aggregates.

(B) They can be tethered by interactions with assemblies of macromolecules outside the cell.

(C) They can be tethered by interactions with assemblies of macromolecules inside the cell.

(D) they can interact with proteins on the surface of another cell.

Protein-Free Lipid Bilayers permeability 

hydrophobic molecules very permeable

small uncharged polar molecules somewhat permeable

large uncharged polar molecules not very permeable

impermeable to ions

intra vs extracellular concentrations of common ions

GREATER OUTSIDE CELL:

Na+, big margin
Mg2+, small margin
H+, small margin
Ca2+, big margin (low concentration both)

GREATER INSIDE CELL:

K+, big margin

two main classes of membrane transport proteins

transporters and channels

Transporters bind the specific solutes and undergo a series of conformational changes

Channels form a pore across the bilayer through which specific solutes can diffuse through

Active Transport Is Mediated by ________ Coupled to an _________

Active Transport Is Mediated by transporters, Coupled to an energy source

Concentration gradient drives passive transport. 

Active Transport is against its concentration gradient and coupled to a source of energy such as ATP hydrolysis or ion gradient. 

For a charged molecule, the electrical potential or membrane potential is also considered. It is defined as electrochemical potential (inside is normally negative).

Coupled transporters

use energy stored in concentration gradients to  couple the downhill transport of one solute to the uphill transport of another

Glucose transport fueled by

a Na+ gradient as symport

Alternates between outward-open and inward-open, and the binding of Na+ and glucose is cooperative

Light and ATP driven pumps

Light-driven pumps couple energy from light to the uphill transport

ATP-driven pumps couple the hydrolysis of ATP to the uphill transport

An _______ Distribution of Transporters in Epithelial Cells Underlies the ___________

Asymmetric Distribution of Transporters in Epithelial Cells Underlies the Transcellular Transport of Solutes

asymmetric distribution of transporters in epithelial cells underlies what process

what is the distribution of transporters in epithelial cells and why do they need a K+/Na+ pump?

uneven distribution underlies transcellular transport of solutes

Na+-linked symporters are located in the apical domains, whereas glucose transporters in the basal or lateral (basolateral) domains passively allow glucose to leave the cells down their concentration gradients.

Why do we need a Na+/ K+ pump?

The Na+ gradient is maintained by an ATP- driven Na+/ K+ pump in the basolateral domains, which keeps the internal concentration of Na+ low —> Na+ symporter uses the gradient to pump in glucose

Three Classes of ATP-Driven Pumps

P-type pumps phosphorylate themselves and maintain gradients of the four ions.

ABC (ATP Binding Cassette) transporters primarily pump small molecules across cell membranes, but also pump large molecules.

V-type proton pumps transfer H+ into organelles such as synaptic vesicles, lysosomes, and plant and yeast vacuoles to acidify their interiors

The Plasma Membrane Na+-K+ Pump Establishes Na+ and K+ Gradients Across__________

The process is _____-genic

across the plasma membrane

A Na+-K+ ATPase antiporter pump acts against their steep electrochemical gradients 

The process is electrogenic as it pumps three positively charged ions out for every two it pumps in. However, it contributes less than 10% for the inside negative membrane potential

ABC Transporters Constitute the __________ Family of Membrane Transport Proteins

largest

● Antimalarial drug chloroquine is pumped out of cell by an acquired ABC transporter of the protist

Ion Channels Are _____ and Fluctuate Between ____ and____ States

Ion channels general characteristics

Types of ion channels

Ion Channels Are Ion-Selective and Fluctuate Between Open and Closed States

• Ions diffuse down their electrochemical gradients through channels

• Ion channels allow 105 times faster rate of transport.

• Ion selectivity is dependent on pore size to atomic dimensions in one particular region.

• The gate is not always open but gated by voltage, ligand, and mechanical forces (hair cells)

• Ligand-gated includes transmitter- gated, ion-gated, or nucleotide-gated

The Function of a Neuron Depends on ________

Neuron structure

its elongated structure

• Dendrites branch from the cell body like antennae to receive signals from the axons of other neurons.

• One long axon conduct signals away from the cell body. An electrical excitation known as an action potential or nerve impulse can travel without attenuation at speeds of 100 meters per second or more.

• Axon branches pass messages to dendrites of other neurons or many target cells such as muscle or gland cells

Voltage-Gated Cation Channels Generate Action Potentials in __________ cells

electrically excitable

• Voltage-gated Na+ channels  open following membrane depolarization from its resting value of -65 mV to about +50 mV in half of a millisecond.

• Na+ channels then automatically inactivate and voltage-gated K+ channels open to restore the normal membrane potential

Transmitter-Gated Ion Channels Convert _________ Signals into _________ Ones at Chemical ______

Chemical. Electrical, at synapses

The _________ Receptors at the Neuromuscular Junction Are __________ Channels

AChR has two __ subunits and three other subunits

Two acetylcholine bind to alpha-subunits, causes:

acetylcholine, excitatory transmitter-gated cation channels

• The acetylcholine Receptor has two alpha and three other subunits. The gate is made by the hydrophobic side chains of five leucine amino acids

• When two acetylcholines bind the two alpha-subunits, a conformational change opens the channels for the through-traffic of Na+ and K+, together with some Ca2+

Neuromuscular Transmission Involves the Sequential Activation of _____ Different Sets of Ion Channels

FIVE different sets

All Eukaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles:

Rough ERs synthesize proteins and lipids, and send them to Golgi apparatus.

Golgi apparatus modifies the proteins and lipids, and dispatches them to various destinations.

Lysosomes contain digestive enzymes to degrade intracellular organelles, and macromolecules and particles from outside through endocytosis. On the way to lysosomes, the materials must pass endosomes.

Peroxisomes are small vesicular compartments that contain enzymes used in various oxidative reactions

Evolutionary Origins Explain the Topological Relationships of Organelles

enclosure of bacterial symbiont by archaeal membrane fusion —> escape of endosymbiont into cytosol —> elaboration of internal compartments

The organelles in the secretory and endocytic pathways have an interior or lumen that is topologically equivalent to the exterior of the cell, including ER, Golgi, endosomes, lysosomes, and peroxisomes

The nuclear membrane may originate from

an invagination of the plasma membrane and pinch off with a double membrane.

The lumen of the ER is _________ with the space between the inner and outer nuclear membranes, and the space is topologically ________ to the extracellular space.

The lumen of the ER is continuous with the space between the inner and outer nuclear membranes, and the space is topologically equivalent to the extracellular space

The nucleus and the cytosol communicate through_______ and topologically __________

The nucleus and the cytosol communicate through nuclear pore complexes and topologically continuous.

4 different ways proteins can move between compartments

Gated transport involves nuclear pore complexes

Protein translocation uses transmembrane protein translocators

Vesicles loaded with a cargo of molecules bud or pinch off, travel, and fuse to a second compartment of topological equivalence

Engulfment refers to moving proteins from the cytosol into the lysosome in autophagy or enclosing chromosomes inside the nucleus during nuclear envelope re-formation after mitosis

protein translocations

nucleus to cytosol: gated transport and engulfment

out of cytosol: protein translocation

everything else pretty much: vesicular transport

Sorting Signals and Sorting Receptors Direct Proteins to the correct _______ __________

cell address

common sorting signals

import into nucleus: positively charged AA

Export from nucleus, Import into ER: hydrophobic AA

import into mitochondria: hydrophobic AA alternating with + charged AA

import into plastid: uncharged polar or hydroxylated AA

Import into peroxisomes: uncharged polar/pos charged/hydrophobic AA at C-terminus

Return to ER: some negatively charged AA

A __________ Directs the ER Signal Sequence to a __________ in the Rough ER Membrane

A Signal-Recognition Particle (SRP) Directs the ER Signal Sequence to a Specific Receptor in the Rough ER Membrane

The hydrophobic ER signal sequences are usually recognized by SRP. SRP receptor binds to SRP-ribosome complex and brings it to the translocator.

The translocator then binds ribosome, inserts the polypeptide chain into the membrane, and transfers it across the lipid bilayer to lumen

is a co-translational translocation

Transmembrane Proteins Contain __________ Segments That Are Recognized Like _____

hydrophobic segments recognized like signal sequences

orientation of hydrophobic segments of multipass transmembrane proteins

interpret context to find orientation

When the next transmembrane segment emerges from ribosome, it inserts into lateral gate of Sec61 in orientation opposite to that of first transmembrane segment

Most Proteins Synthesized in the Rough ER Are Glycosylated by the Addition of a____________

Common N-Linked Oligosaccharide

A) An oligosaccharide is attached to asparagines in the sequences Asn-X-Ser or Asn-X-Thr (where X is any amino acid except proline). The five sugars in gray box form the core region that survive extensive trimming in the Golgi apparatus.

B) A lipid molecule anchors the precursor oligosaccharide in ER lumen membrane, which is then transferred to Asn by the enzyme

Sorting Signal Sequences for import into peroxisomes

import receptor recognizes the Ser-lys-leu signal in C-terminus of proteins made in cytosolic ribosomes

Needs ATP

mitochondrion v chloroplast image

Thylakoid membrane is not connected to the inner membrane.

Transport Into the Inner Mitochondrial Membrane Occurs Via Several (4) Routes:

(A) A hydrophobic transmembrane segment binds to TOM and then TIM23 which puts it across the membrane

(B) Binding of the proteins to the chaperones guides to TIM22 complex, which then insert the multipass inner membrane proteins.

(C) The protein is first made in the matrix space, and a signal then directs it to the inner membrane through OXA complex.

(D) Nuclear-encoded proteins translocate into the matrix space via the TOM and TIM23 complexes. Cleavage of the signal sequence unmasks an adjacent hydrophobic signal sequence at the new N- terminus. It is inserted to the inner membrane visa OXA complex

Two Signal Sequences Direct Proteins to the ___________ in Chloroplasts

Two Signal Sequences Direct Proteins to the Thylakoid Membrane in Chloroplasts

A. Chloroplast signal sequence initiates the translocation through TOC then TIC (GTP or ATP dependent) then peptide is in stroma. Cleavage unmasks the thylakoid signal sequence that initiates the translocation across the thylakoid membrane.

B. Translocation to the thylakoid space or membrane by using:

1) homolog of Sec61 (multipass proteins) that mediates protein translocation across the bacterial plasma membrane

2) homolog of OXA pathway (also using SRP homolog)

3) a TAT (twin arginine translocation) pathway, arginines important for directions

protein import into chloroplast organelles evolved from

a bacterial protein-export system

Plant cells: TIC/TOC

Bacterium: Bam/Tam

TIC —> TamB
TOC → TamA/BamA

nuclear envelope structure

Nuclear Envelope (inner + outer nuclear membrane) perforated by nuclear pore complexes (NPCs)

Nuclear Localization Signals Direct Nuclear Proteins to the

____

nucleus

mutation of nuclear import signal can lead to greater localization

Nuclear Import Receptors Bind to Both _______ _____ _________ and ______

also sometimes can bind ___ repeats in unstructured domains of _______ ____________

Nuclear Import Receptors Bind to Both Nuclear Localization Signals and NPC Proteins

Nuclear import receptors also bind the phenylalanine-glycine (FG) repeats in the unstructured domains of the channel nucleoporins

The Ran GTPase Imposes _________ on Transport Through NPCs

directionality

Ran-GDP is in the cytosol and Ran-GTP is in the nucleus. It is due to the activity of GTPase activating protein (GAP) in the cytosol and guanine exchange factor (GEF) in the nucleus

nuclear export vs import

export works just like import but in reverse

For nuclear import, Ran-GTP binding causes the receptor to release the cargo in the nuclear side, critical for the directionality of the nuclear transport.

For nuclear export, Ran-GTP binding promotes the loading of cargo. GAP in the cytosol triggers GTP hydrolysis and its dissociation from the receptor

During Mitosis the Nuclear Envelope _________

disassembles

Nuclear lamina is a meshwork made by protein complex of lamins and gives shape and stability of the nuclear envelope.

During mitosis, the NPCs and lamina disassemble and the nuclear envelope fragments

Two different structural features for inside and outside membrane protein domains:

Most membrane proteins in animal cells are glycosylated on the cell surface, or the oligosaccharides are only found on non-cytosolic surface

The intrachain or interchain disulfide bonds are only found on non-cytosolic surface. The sulfhydryl group is not forming disulfide bond in the cytosolic side due to a reducing environment.

Channels always mediate _____ transport, whereas transporters mediate either ____ or ____ transport. 

channels = always passive
transporters = active or passive

epithelial cell domains

basal domain = at base

lateral domain = middle of cell

apical domain = end of cell, tip

ABC transporters in bacteria vs eukaryotes

Both importers and exporters are found in bacteria. 

In eukaryotes,  most ABC transporters are exporters for ions, amino acids, polysaccharides, lipids, drugs, and peptides from cytosol to the extracellular space or into ER (similar topology as outside), or from the mitochondrial matrix to the cytosol (much like bacteria).