Final exam Flashcards
What are the four classes of macromolecules?
Proteins, lipids, carbohydrates, nucleic acids
The ___________ structure of proteins consists of the protein’s amino acid sequence.
Primary
Secondary protein structure consists of alpha helices and beta-pleated sheets that form from ____________ between the backbones of amino acids, not side chain interactions.
Hydrogen bonds
Super-secondary secondary structures are structures that form from secondary structures grouped in specific ways; they are also called _____________.
Motifs
A ___________________ is a similar 3D structure conserved among proteins that serve a similar function.
Motif
Tertiary structures of proteins result from ______________ interactions: (1) hydrophobic interactions, (2) ionic bonds/salt bridges, (3) hydrogen bonds, and (4) _________________ between sulfhydryl groups of cysteine residues.
Side-chain or R-group interactions
Disulfide bridges (covalent interaction)
___________________ structure occurs when a protein is composed of more than one polypeptide chain.
Quarternary
What are stable, independelty folding, compact structural units within a protein that have relative independent structure and function distinguishable from other regions and stabilized through the same kind of linkages at the tertiary level?
Domains
Why do disulfide bridge not exist in the cytosol?
They are highly oxidized, but the cytosol is highly reduced
How are proteins regulated?
Quantity, activity, location
What are the ways in which gene expression can be regulated?
Transcriptional control
RNA processing control
RNA localization
Translational control
Post-translational control
mRNA degradation
Protein-activity control
What primarily mediates transcriptional control of gene expression?
Transcription factors
Trans-acting factors or ___________________ factors bind cis-regulatory elements and work to speed up or slow down transcription.
Transcription factors
Transcriptional _______________ bind to cis-regulatory sequences and turn genes off.
Repressors
Transcriptional ________________ bind to cis-regulatory sequences and turn genes on.
Activators
_________________ and ______________ bind to activators and repressors, not DNA, and assist in gene regulation.
Coactivators
Corepressors
What are two mechanisms through which transcriptional activators function?
Chromatin remodeling
RNA polymerase recruitment
General transcription factors and RNA polymerase cannot displace nucleosomes on their own; therefore transcription activator promote transcription by triggering changes to chromatin structure near promoters, making the DNA more accessible. Transcription activators do so via four processes: (1) covalent histone modification, (2) _________________ remodeling, (3) nucelosome removal, and (4) histone replacement.
Nucleosome
What are four ways in which activators trigger chromatin remodeling?
Covalent histone modification
Nucleosome remodeling
Nucleosome removal
Histone replacement
Transcription activators can act on RNA polymerase in four ways. What are they?
Promote binding of additional regulators
Recruit RNA polymerases to promoters
Release RNA polymerase to begin transcription
Release RNA polymerase from pause
Transcription activators can promot binding of additional regulators, recruit __________________ to promoter region, release _______________ to begin transcription, and release _______________ from pause (all blanks are the same answer).
RNA polymerase
Genes encoding the most important developmental regulatory proteins are kept tightly repressed or activated?
Repressed
What are six mechanisms of transcription repression?
Competitive DNA binding
Masking the acitvation surface
Direct interaction with general transcription factors
Recruitment of chromatin remodeling complexes
Recruitment of histone deacetylases
Recrtuitment of histone methyl transferases
____________________ of DNA usually increases transcription whereas _____________ of DNA usually decreases transcription.
Acetylation
Methylation
What is combinatorial control?
Multiple activators or multiple copies of a single activator work synergistically to initiate transcription
What is mean by transcriptional synergy?
Transcription factors often exhibit synergy, meaning that they work together to produce a transcription rate much higher than the sum of their transcription rates working alone
________________ refers to the maintenance of cell differentiation through subsequent cell generations.
Cell memory
The early Drosophila embryo is a __________________, meaning it has multiple nuclei in a shared cytoplasm.
Syncytium
The position of transcriptional factors is crucial for Drosophilia development. Bicoid and Hunchback are localized to the ___________ of the cell while Giant and Kruppel are not.
Anterior
Bicoid and Hunchback are transcription _______________ while Giant and Kruppel are transcription _____________.
Activators
Repressors
Nuclei in the syncytium begin to express different genes because they are exposed to different __________________ that are localized to specific areas of the cell.
Transcription regulators
To what does Eve refer?
Even-skipped gene, which is important in body patterning in Drosophilia flies
Bicoid, Hunchback, Kruppel, and Giant are transcription regulators of Eve expression in which stripe?
Stripe 2
If Bicoid and Hunchback are inactivated, what happens to stripe 2?
No stripe 2 develops because Bicoid and Hunchback are activators
If Kruppel and Giant are inhibited, what happens to stripe 2?
Stripe 2 is diffused throughout the cell because Kruppel and Giant are repressors
What are three types of RNA processing discussed in class?
5’ cap of 7-methylguanosine
3’ cleavage and polyadenylation
Alternative splicing
Alternative splicing allows for the production of different ______________ from a single gene.
Proteins
Is alternative splicing uniform across cell types and developmental stages?
No; alternative splicing may differ in different cell types, in a single cell type (due to extracellular signals), or in a single cell type during different stages of development
RNA splicing removes __________ sequences from newly transcribed pre-mRNAs.
Introns
A mRNA molecule only becomes designated as such after which two actions occur?
Addition of 5’ 7-methylguanosine cap and 3’ cleavage and polyadenylation
Although alternative splicing is energy inefficient, it is proposed that over time, RNA splicing increases ___________________ in organisms and for this reason it has continued.
Genetic variability
___________________ sequences signal where splicing occurs.
Nucleotide
RNA splicing is performed by the ____________.
Splicesome
ATP is required for assembly and rearrangement of the _________________.
Splicesome
Each __________ codes for one domain.
Exon
A domain is the product of a single _____________.
Exon
A protein _____________ is a substructure produced by any contiguous part of a polypeptide chain that can fold independently of the rest of the protein into a compact, stable substructure.
Domain
The _______________ protein is a tyrosine-kinase with three domains: ____________, ___________, and ____________.
Src
SH1
SH2
SH3
SH1 is the _______________ site; SH2 is the _________________ binding site; and SH3 binds proline-rich regions of other proteins.
Enzymatic site
Phosphotyrosine-binding site
Domains are highly _____________________.
Conserved
_______________________ _______________________ during evolution has resulted in domain replication and shuffling.
Genetic recombination
Some domains are found in many different proteins. These serine proteases share the same catalytic domain. Differential __________ is conferred by other domains.
Regulation
Only about _____% of all synthesized RNA ever leaves the nucleus.
5
What degrades improperly processed RNA molecules in the nucleus?
The nuclear exosome
As RNA is processed, it loses certain proteins; only when specific proteins are present including _______________________________ (hnRNPs) that a mRNA molecule is exported from the nucleus.
Heterogeneous nuclear ribonuclear proteins
Successfully produced mRNAs are guided through _________ ____________ _____________, aqueous channels in the nuclear membrane that directly connect the nucleoplasm with the cytosol. The cell uses _______ to move mRNAs through these complexes. This transport occurs via _________ _________ _________, which are attached to mRNA and dissociate once mRNA has left the nucleus.
Nuclear pore complexes
ATP
Nuclear transport receptors
What are three ways in which mRNA can be localized in the cell?
Directed transport on the cytoskeleton
Random diffusion and trapping
Generalized degradation in combination with local transport by trapping
Where are signals for mRNA localization located?
The 3’ untranslated region (UTR)
Three distinct mechanisms account for the asymmetric distribution of mRNAs within cells: (1) localized _____________ from degradation, (2) diffusion-coupled local ______________, and (3) directed transport along a ________________ cytoskeleton.
Protection
Entrapment
Polarized
mRNA is localized in spatially distinct patterns depending upon the needs of the cell. In most cell types, we find our microfilament structures near the cell membrane in a region called the ___________. In some cell types, notably ____________, there is an even high concentration of actin, specifically beta actin, and its mRNA near the cortex. Fibroblasts are cells in connective tissue that produce collage and other fibers, and in these cells, beta actin mRNA localizes to the _________________, where translation is required for cytoskeleton-mediated motility.
Cortex
Fibroblasts
Lamellipodia
Fibroblasts must be able to move to replair connective tissue, which is why the “foot layer” or ________________________ is important.
Lamellipodia
What experiment did this photo demonstrate?
The experiment demonstrated that the chimeric mRNA was concentrated in the leading edge of the fibroblast, supporting the idea that mRNA is localized for translation.
Experiment: Actin gene was cloned into an expression vector, which produced actin-beta-galactosidase chimeric mRNA and protein. The gene was transfected into chick embryo fibroblasts, and in situ hybridization with beta-galactosidase-specific probes confirmed mRNA accumulates in the lamellipodia just like the protein.
What did this experiment demonstrate?
The two short regions of the beta-actin gene, both of which were located in the 3’ untranslated region, were critical for mRNA localization. One of the regions - the proximal region - was a stronger driver of localization than the distal region.
What is meant by a beta-actin zipcode?
The zipcode refers to two consensus sequences found by scientists that ultimately led to mRNA localization
What is the zipcode-binding protein?
A protein with a gene sequence that shares two highly conserved hnRNP (heterogenous nuclear ribonuclear protein) and nuclear export sequences (NES)
It is currently believed that _____________________ underlie the mechanism for beta-actin localization.
Microfilaments
In Drosophilia development, egg ____________ is determined by localized gene expression in the oocyte and early embryo.
Polarity
_________________ is a morphogen transcription factor at the anterior end of the Drosophilia embryo. ____________ is a morphogen transcription factor at the posterior end.
Bicoid
Nanos
A mutation in Bicoid results in flies with ___________. A mutation in Nanos results in flies with ____________.
Two tails
No abdomens
What facilitates Nanos mRNA localization?
Oskar
Bicoid mRNA localization requires binding by multiple binding proteins including _________ __________ and ____________.
Motor proteins
Microtubules
Nanos mRNA is initiatially __________ in the oocyte cytoplasm but remains translationally repressed, except in the ____________ end, where translation repression is released and mRNA degradation fails.
Diffuse
Posterior
Nanos localization requires a ________________ ______________ ___________ in the 3’ untranslated region.
Translational control element (TCE)
Without the translational control element, ____________ mRNA does not localize and does not ____________, remaining diffuse throughout the cytosol.
Nanos
Degrade
What did researchers determine binds to the translational control element of Nanos, inhibits translation, and prevents localization?
Smaug
Assays for poly-A tail length showed that degradation of Nanos requires ______________ binding to the 3’ ___________.
Smaug
3’ UTR
Smaug represses translation of Nanos and recruits a __________________.
Deadenylase
Both ___________________ and the _____________________ (TCE) are required for translation repression and degradation of Nanos.
Smaug
Translational control element (TCE)
Smaug was found to be the protein that binds to the TCE of Nanos, which represses Nanos ________________ and recruits a ___________, a ribonuclease, to degrade Nanos mRNA.
Translation
Deadenylase
Nanos is selectively translated at the posterior pole by binding to _____________.
Oskar
How does Oskar protect Nanos mRNA?
It beings the TCE and prevents Smaug from binding, thereby preventing degradation and releasing translational repression
Oskar mRNA is localized to the ______________ pole by binding of _________________ (RNPs) and kinesin-1-dependent transport along _______________.
Posterior
Ribonucleoproteins
Microtubules
What does RNPs stand for?
Ribonucleoproteins
Which motor protein is responsible for Oskar localization?
Kinesin-1
What are four ways in which translation can be regulated?
- The protein product of a mRNA performs regulatory action on its own mRNA
- A stem-loop structure can prevent movement of the ribosome
- A small molecule can bind stem-loop structure
- Antisense RNA
Iron regulates the expression of certain genes. ___________ is an iron-binding protein that binds to and holds on to iron in cells until needed.
Ferritin
Ferritin mRNA can be bound by a protein that inhibits its translation. This protein is ____________ _____________ or the ________________ ______________ _______________ (IRE) binding protein.
Cytosolic aconitase
Iron Response Element (IRE) binding protein
The Iron Response Element (IRE) forms a stem-loop structure that is stabilized by _____________________.
Cytosolic aconitase or IRE binding protein
When iron levels in the body increase, iron attaches to the _________________________, causing a conformational change that ultimately releases the protein from the IRE sequence.
Cytosolic aconitase or IRE binding protein
Iron binding to cytosolic aconitase or IRE binding protein induces a conformational change that releases the protein from the IRE sequence and destabilizes the __________________ structure. This ultimately enables the translation of ____________, which can bind to and store increased iron in the cell.
Stem-loop structure
Ferritin
What is transferrin?
A blood protein that transports iron throughout the cell
When iron levels are low, what happens to transferrin receptors?
They are upregulated so that more iron can be brought into the cell
Normally, the IRE binding protein binds the stem-loop structure at the 3’ UTR of the transferrin receptor mRNA, preventing mRNA degradation. However, when iron levels are ________, there is no longer a need for transferrin receptors, so iron binds the IRE binding protein, releasing it from the 3’ UTR. This leaves the _____________ structure open to degradation.
High
Stem-loop structure
During iron starvation, the binding of ______________ or the IRE binding protein to the 5’ UTR of ___________ mRNA blocks translation initiation; its binding to the 3’ UTR of the ________ receptor mRNA blocks an endonuclease cleavage site, thereby stabilizing the mRNA. In this way, _______ is downregulated and _______ receptors are upregulated, bring iron into the cell.
Cytosolic aconitase
Ferritin
Transferrin
Ferritin
Transferrin
When iron levels are low, we want to _____________ ferritin levels and increase _________________ receptors.
Decrease
Transferrin
In response to high iron concentrations, ______________ synthesis is increased. Iron binds to _________________ or the IRE binding protein in the 5’ UTR, which releases from the mRNA and enables translation. Iron binding also prevents ______________ or the IRE binding protein from binding to the stem-loop structure in the _______, thereby exposing an ________________ cleavage site and enabling degradation of __________________ mRNA.
Ferritin
Cytosolic aconitase
Cytosolic aconitase
3’ UTRE
Endonucleolytic
Transferrin receptor
Be able to explain this figure.
Control of mRNA degradation can be regulated by _________ RNAs, like ___________ (miRNAs), ____________________ (siRNAs), and _______________________ (piRNAs).
Noncoding
MicroRNAs
Small interfering RNAs
Piwi-interacting RNAs
microRNAs (miRNAs) regulate mRNA _______ and stability. They are synthesized by RNA polymerase II, undergo capping and polyadenylation, and assemble with ___________ to form the ___-__________ ____________ ____________ (RISC).
Translation
Argonaute
RNA-induced silencing complex
________ _________ __________ are short double-stranded RNA molecules that are bound by Argonaute and other RISC components to degrade mRNA.
Short interfering RNAs
________ _________ _____ are made in the germ line and block the movement of transposable elements
Piwi interacting RNAs (piRNAs)
What 7 covalent modifications occur in cells that regulate proteins post-translationally?
- •Proteolysis
- •Glycosylation
- •Phosphorylation
- •Acylation
- •Acetylation
- •Methylation
- •Protein tags
Many proteins are synthesized in larger forms known as “pro.” Many enzymes are are also synthesized in this way (i.e., “ogen”). These larger forms are _______ and require modification by _________ to engage biological functioning. A well-known example of this post-translation modification is __________.
Insulin
What is glycosylation?
is the process of adding carbohydrate groups onto proteins
There are ______ types of glycosylation depending upon where and how the carbohydrate is attached: (1) ___-linked glycosylation occurs when the carbohydrate group is attached to the _______ atom on the R group of asparagine, and (2) ___-linked glycosylation: carbohydrate group is attached to the oxygen atom on the R group of __________ or ________.
Two
N-linked
Nitrogen
O-linked
Serine or threonine
Are glycosylated proteinscommonly found in the cytosol?
No, they are most often found in proteins destined for secretion
Proteins that recognize and bind carbohydrate structures are called __________.
Lectins
____________ are a type of lectin that is important in cell-cell recognition and communication, immune system function, and brain development
Selectins
Glycosylation influences protein __________, inhibits __________, provide binding sites for lectins, and function in cell-cell recognition.
Folding
Degradation
Lectins
Which three amino acids can be phosphorylated?
Serine
Threonine
Tyrosine
What enzymes phosphorylate molecules?
Kinases
What enzymes dephosphorylate molecules?
Phosphatases
Most enzymes that can phosphorylate serine can also phosphorylate threonine; these kinases are called _________ _____________ kinases.
Serine-threonine kinases
Only ___________ kinases can phosphorylate tyrosine.
Tyrosine kinases
What is the purpose of phosphorylation and dephosphorylation in the cell?
The purpose of phosphorylation and de-phosphorylation is for activation and deactivation; when a protein is phosphorylated or dephosphorylated, conformational changes occur, which will lead to either an activating or deactivating change
Proteins can be acylated by the addition of _________ _________, like myristate and palmitate, and __________ ________, like farnesyl and geranylgeranyl.
Fatty acid chains
Isoprenyl chains
Why is the addition of acyl chains important?
They anchor and localize proteins to the plasma membrane
Isoprenyl groups are ____ carbon molecular units that are also called __________. They can be linked together to form longer chains. When two are linked together, a ____ carbon unit is formed and called a _________. When three are linked together, a ___ carbon unit is formed and called a ________. And when four are linked together, a 20 carbon unit is formed and called a geranylgeranyl.
Five
Terpenes
10 carbon
Geranyl
15 carbon
farnesyl
Acetylation and methylation most often occur on histones; _________ groups are always added to __________ residues, and _________ groups are usually, but not always, added to ___________residues, too
Acetyl groups
Lysine
Methyl groups
Lysine
________ acetyl transferases and deacetylases as well as _______ methyl transferases and demethylases are enzymes recruited by transcription factors that recognize specific DNA sequences
Histone
Histone
The amino tails of ____________ can be heavily modified, and modifications usually occur on lysine residues. They may differ throughout the genome and may change during the life of the cell.
Histones
What is the histone code?
a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends
What two protein tags did we discuss in class?
Ubiquitin
SUMO (small ubiquitin-related modifier)
___________ is a highly conserved 76 amino acid protein that is found in a huge range of organisms and remains nearly identical across them. It is conjugated by its C terminus to an internal _______ residue of the target protein
Ubiquitin
Internal lysine (K)
_________ is a pattern of _____ubiquination in which one ubiquitin attaches to an internal ______ of the target protein and additional ubiquitins are added to the 48th lysine residue of the first ubiquitin; ______ is similar, except each additional ubiquitin is added to the 63rd lysine on the previous ubiquitin molecule
Lys48
Polyubiquination
Lysine
Lys63
Ubiquitination of a protein involves three proteins: ____, ____, and _____.
E1
E2
E3
In the process of ubiquination, ______ is the ubiquitin-activating enzyme. E2 is the ___________; together they complex together, and _____ transfers its ubiquitin to _____,
E1
Ubiquitin-conjugating enzyme
E1 transfers to E2
_____ stores the ubiquitin until E3 or ________ and a target protein arrive; once E3 and the target protein are in the vicinity, _____ facilitates the movement of ubiquitin to the target protein
E2
Ubiquitin ligase
E2
What is the role of E3 in ubiquination?
E3 simply serves to recognize the target proteins in need of ubiquitination and is the site of substrate binding
Be familiar with the general process of ubiquination.
______ubiquination on histones is part of the histone code. ______ubiquination, specifically __ ___, on histones and monoubiquination on many other proteins, are common at sites of DNA repair
Multiubiquination via mono, di, or poly (via K63) on a transmembrane protein can trigger ________ and trafficking to the _________for ___________.
_____ubiquination (__ __) marks proteins for proteosomal degradation
Monoubiquination
Polyubiquination K63
Endocytosis
Lysosome
Degradation
Polyubiquination K48
K63 polyubiquination is involved in ____ ______ and _______________ of transmembrane proteins that are trafficked to the lysosome for degradation. K48 polyubiquination is involved in ______ ________.
DNA repair
Endocytosis of transmembrane proteins
Proteosomal degradation
SUMOylation is involved in four things. What are they?
–DNA damage repair
–Chromatin organization & transcription
–Chromosome segregation & cytokinesis
–Nuclear protein import
Covalent modifications are reversible but require a great deal of energy and are therefore reserved for “one-and-done” events. _____-__________ modifications do not require such an energy expenditure and can include interactions with other proteins and __________ proteins, like cAMP, calcium ions, and GTP.
Non-covalent
Regulatory
Which occurs more frequently in the cell? Covalent or non-covalent modifications?
Non-covalent
Non-covalent binding can result in three things. What are they?
- Formation of structural complexes
- Localization of a protein within the cell
- Regulation of a protein’s activity
____________ binding is mediated by weak intermolecular forces
Reversible
_________________ interactions are not considered intermolecular forces although they are often included under the umbrella term.
Hydrophobic interactinos
Calcium can directly bind to and regulate other proteins. It can also bind to _______-_________ _______, which then binds to and regulates other proteins. The most common is _________, which requires two or more calcium ions to bind for activation.
Calcium-binding proteins (CBPs)
Calmodulin
G-proteins are intrinsic _______ases.
GTPases
Proteins that bind to GTP are called GTP-binding proteins, commonly called G-proteins. When bound to GTP, the protein is usually _______. When bound to GDP, the protein is usually ________.
Activated
Inactivated
There is a great deal of ____ in the cell. High-energy nucleotides are not scarce.
GTP
Binding to _____ to a G-protein causes a ______________ change, leading to its activation
GTP
Conformational change
How are G-proteins regulated?
GEFs and GAPs
What does GEF stand for?
Guanine nucleotide exchange factor
What does GAP stand for?
GTPase activating protein
What does GEF do?
Assists in releasing GDP from G-proteins; GEF binds to the G-protein, causes a conformational change in the G-protein, which decreases the G-protein’s affinity for GDP, thereby releasing GDP; this conformational change simultaneously increases the G-protein’s affinity for GTP
What does GAP do?
Binding of GAP causes a conformational change in the G-protein that increases the catalytic activity of the G-protein, causing it to hydrolyze its GTP faster
What hydrolyzes the GTP? The G-protein or its GAP?
The G-protein
G-proteins regulate many activites in the cell. For example, ___________ is actually a modified form of G-proteins!
Tubulin
There are two ways to degrade a protein in the cell. What are they?
Degradation via the lysosome
Degradation via proteosomes (protein-specific)
_____________ contain acid hydrolases because these enzymes only function at a lower pH – lower in comparison to blood and cytosol – usually around pH ____. These hydrolases can target nearly any type of molecule within the cell
Lysosomes
pH5
Lysosome membranes have ______ ___________ ______ to keep the interior acidified
ATPase hydrogen pumps
Why must the lysosome have an acidic enviroment?
Protection; if the lysosome bursts and its contents spill into the cytosol, nothing damaging will occur since the hydrolases are only active at lower pHs; they will immediately denature in the more basic environment of the cell
What is autophagy?
Autophagy is a self eating process cells go through for normal organelle turnover; mitochondria are especially prone to becoming worn out and in need of turnover
_________________ are cylindrical multi-protein complexes with hollow inner chamber lined with proteases and multi-protein caps on each end
•
Proteasomes
Where are proteasomes abudant?
Cytosol and nucleus
How do proteosomes work?
- Target proteins are ‘tagged’ with polyubiquitin
- The cap complex contains a ubiquitin receptor, a ubiquitin hydrolase, and unfoldases
- Proteins are unfolded, threaded through the cylinder, cut into short peptides which are released through the opposite end
____________ function as sensors and/or molecular switches which regulate a variety of cellular processes
G-proteins
What are the three stages of translation?
Initiation
Elongation
Termination
What are the 5 steps of translation initiation?
- eIF2-GTP binds tRNA Met and small ribosomal subunit, forming the 43S pre-initiation complex
- Multiple IFs bind mRNA
- 43S complex binds mRNA at 5’ end
- 43S complex scans in 5’ to 3’ direction
- The.Kozak sequence (ACCAUGG) is recognized; GTP is hydrolyzed with eIF5 serves as its GAP. eIF2 and most other eIFs detach prior to 60s subunit binding
eIF__ is a G-protein
eIF2
What recognizes the Kozak sequence?
The 43S Pre-Initiation Complex
What triggers hydrolysis of eIF2’s GTP?
The ribosome
What actually helps eIF2 hydrolyze its GTP? In other words, what is the GAP for eIF2?
eIF5
Initiation factors are ___________.
Reused
Once detached from the 43S pre-initiation complex, eIF2 will release its GDP, which requires its GEF: _________.
eIF2B
What initiation factor plays a global role in translation?
eIF2 plays a really important role as a more global regulator of translation; it is possible to both activate and deactivate eIF2 and thereby control translation
eIF2 can be phosphorylated by ______ kinases, which _________ eIF2 by increasing its affinity for its GEF _________.
eIF2 kinases
Inactivates
eIF2B
A fraction of ________________ eIF2 can trap nearly all of the ______ in the cell.
Phosphorylated
eIF2B
What activates eIF2 kinases?
Stress
What four cell stressors can activate eIF2 kinases in the cell and thereby shutdown translation?
Nutrient limitation, ER stress, viral infection, and heme deprivation
What eIF2 kinase is activated by nutrient limitation?
GCN2
What specifically activates GCN2?
Lack of amino acids
What is structurally important about GCN2 - an eIF2 kinase?
It shares homology with histidyl-tRNA synthetase (HisRS)
Which amino acid is critical to GCN2 activation?
Histidine (specifically its lack)
3AT, an inhibitor of histidine formation, and SM, an inhibitor of branched-chain amino acids, ultimately _________ GCN2 and thereby inactivated eIF2.
Activated via phosphorylation
Where is the connection between low amino acid concentration and GCN2 kinase activation?
GCN2 (eIF2 kinase) binds directly to uncharged tRNA, meaning that when amino acids are unavailable, GCN2 can bind to tRNA and presumably be activated, thereby enabling GCN2 to phosphorylate eIF2 and inhibit translation initiation
What eIF2 kinase is activated under ER stress?
PEK/PERK
Where is PEK/PERK located?
The ER membrane
What specifically triggers PEK/PERK activation?
The unfolded protein response (UPR)
PEK/PERK is normally bound by __________ _________, a member of the HSP 70 family. When unfolded proteins are present; however, this protein undergoes a conformational change and releases PEK/PERK. PEK/PERK then dimerze, crossphosphorylate, and activate the catalytic domains responsible for _____ phosphorylation, which inhibits translation initiation.
Binding Protein (BiP)
eIF2
What does BiP stand for?
Binding Protein
The association of PEK/PERK and BiP (Binding Protein) was demonstrated using an ________________ experiment with ______ _______ cells.
Immunoprecipitation experiment
Pancreatic acinar cells
Researchers demonstrated that under ER stress BiP (Binding Protein) and PEK/PERK dissociate by using ________ (Tg), which inhibits the calcium ATPase in the ER membrane, and ________ (DTT), which is a reducing agent. Tg and DTT trigger ER stress.
Thapsigargin
Dithiothreitol
What eIF2 kinase is activated under viral infection?
PKR
How does PKR recognize viral infection in cells?
Virus-infected cells have a lot of double stranded RNA; PKR possesses a double stranded RNA binding domain
How is PKR activated under viral infection?
PKR recognizes and binds double-stranded DNA viral fragments, which induces a conformational change in PKR that allows it to dimerize with another PKR, crossphosphorylate, and activate each other, thereby enabling PKR to act as an eIF2 kinase and phosphorylate/deactivate eIF2
Why does the cell shut down translation under viral infection?
Viruses replicate using the same machinery of the cell; therefore stopping all translation inhibits viral replication and the creation of more viral proteins
Hemoglobin synthesis requires _________.
Heme
What eIF2 kinase is activated under heme deprivation?
Heme-controlled inhibitor (HCI)
What does HCI stand for?
Heme-controlled inhibitor
When is heme-controlled inhibitor activated?
When there is a lack of heme needed to make red blood cells
When heme is present, it binds to the ____-________ _______ and deactivates it, turning off its kinase activity and preventing it from functioning as an eIF2 kinase.
Heme-controlled inhibitor
When the the heme-controlled inhibitor activated?
When there is no heme bound to it
In addition to the 5 steps of initiation previously discussed, there are two more steps. What are they?
- 60S ribosomal subunit binds along with eIF5B-GTP to form 80s initiation complex
- GTP is hydrolyzed only after large and small subunits join. A rRNA serves as the GAP likely in small subunit and requires eIF1A. eIF5B detaches before translation begins
What must detach from the A site of the ribosome for translation to begin?
eIF5B
If the G-protein is a sensor for some event and must hydrolyze its GTP before translation can proceed, what is it sensing?
The correct binding of the large and small ribosomal subunits
eIF__-GTP binds tRNA (Methionine) and the samll ribosomal subunit, mediating the formation of the 43S Pre-Initiation Complex. eIF__ serves as the GAP for eIF__. These early initiation factors then dissociate, allowing for the 60S ribosomal subunit and eIF__ __-GTP to for the 80S Initiation Complex. rRNA in the small riibosomal subunit serves as the GAP but requires eIF__ __ to do so. eIF__ __ must detach from the ribosome before translation can begin.
eIF2-GTP
eIF5
eIF2
eIF5B-GTP
eIF1A
eIF5B
eEF__ __-GTP shepherds each tRNA into the A site for elongation. Proper codon:anticodon binding leads to conformational changes in the large ribosomal subunit (rRNA serves as GAP), which triggers GTP hydrolysis by eEF__ __, which then releases the tRNA and detaches before peptide bond formation. eEF__ __ serves as the GEF for eEF__ __.
eEF1alpha
eEF1alpha
eEFbeta-gamma
eEF1alpha
There is __ ________ in bringing tRNAs to ribosome; it is just _______ diffusion
No selection
Random diffusion
For translocation of the ribosome to occur, what elongation factor is required?
eEF2-GTP
Binding of eEF__-GTP causes a counter-clockwise rotation in the small ribosomal subunit that (1) destabilizes the ribosome-tRNA interaction, (2) widens the channel openings for the mRNA, and (3) triggers GTP hydrolysis followed by eEF_ detachment. The ribosome possesses the GAP activity, but we do not know where. Once eEF__ detaches, the small subunit rotates clockwise back to its original conformation
eEF2
eEF2
eEF2
What is the mechanism of translocation likened to?
Ratchet
When the ribosome recognizes a stop codon in the A site, release factors bind, catalyzing the addition of a water molecule to cleave the bond between the polypeptide chain and the tRNA molecule. eRF__ interacts with and binds to stop codons. Its binding brings eRF__-GTP closer to the ribsome. eRF__ is the G-protein. Once hydrolyzed, both eRF__ and eRF__ detach. This detachment triggers the recruitment of water to the ribosome.
eRF1
eRF3-GTP
eRF3
eRF1 and eRF3
What triggers hydrolysis of the peptide bond and tRNA molecule during translation termination?
The detachment of eRF1 and eRF3 from the stop codon
Experiments using __ ___ ____________ and ________________ demonstrated that GTP hydrolysis of eRF3 can only occur when eRF1 and the ribosome were present.
Thin-layer chromatography
Phosphoimaging
The nucleus is studded with hundreds of _________ _________ ______________, which are large assemblies of proteins, that form channels through the double phospholipid bilayer that makes up the nuclear membrane.
Nuclear pore complexes
Proteins less than ___ kDa can diffuse freely through the nuclear pore complex, but anything larger than ___ kDa must be “actively” transported.
40 kDa
40 kDa
The interior of the nuclear pore complex is composed of amino acids ____________ and _________ repeats, both of which are hydrophobic and impede the movement of hydrophilic materials through the pore.
Phenylalanine
Glycine
Proteins to be imported into or exported out of the nucleus have ___________ ___________ sequences
Consensus signal sequences
Proteins to reside within the nucleus bear _________ __________ ____________.
Nuclear localization signals
Nuclear localization signals usually have a series of three or more _____ and _____ residues.
Lysine
Arginine
What recognizes nuclear localization signals?
Importins
__________ are a family of proteins that bind cargo and shepherd these cargo through the nuclear pore complex, navigating the FG repeats inside the pore
Importins
Proteins and some RNAs to be exported from the nucleus have ________ ________ ____________.
Nuclear export signals
Nuclear export signals were first discovered in which disease protein?
HIV protein reverse transcriptase
Nuclear export signals are usually high in which amino acid?
Leucine
Nuclear export signals are recognized and bound by ________.
Exportins
Importins bind cargo with nuclear localization signals and these importins diffuse through the nuclear pore complex. Once inside the nucleus, importins interact with the G-protein, ____, which is in its GTP bound state. Binding of the importin to the G-protein causes a conformational change in importin that decreases its affinity for its cargo. Importin and ____-GTP remain complexed together and diffuse ____ of the nucleus. Once in the cytosol, ___-GTP encounters its GAP, ___-GAP, and releases importin.
Ran-GTP
Ran-GTP
Out of
Ran-GTP
Ran-GAP
Exportins bind cargos with nuclear export signals as they simultaneously bind the G-protein, ____, in its GTP state. This triad diffuses out of the nucleus through the nuclear pore complex. Once in the cytosol, ___-GTP interacts with its GAP, ___-GAP, hydrolyzing its GTP to GDP and releasing exportin and its cargo.
Ran
Ran-GTP
Ran-GAP
Nuclear transport is a form of _________ transport because molecules are being moves _____ their concentration gradients
Active
Against
Where does the energy come from to drive nuclear transport?
Only cargo proteins are moved against their concentration gradients; cargo transport is coupled with Ran movement, which moves down its concentration gradients
The directionality of nuclear transport is depended solely on the relative concentration gradients of ________ and ______ across the nuclear membrane
Ran-GTP
Ran-GDP
Ran-GTP is higher inside or outside the nucleus?
Inside
The cell takes advantage of the relative concentrations of Ran by coupling the movement of other molecules against their concentration gradients. In the cytosol, ________ binds to cargo and moves down its own concentration gradient by entering the nucleus. Once in the nucleus, _____ binds Ran-___, which triggers cargo release and returns ________ to the cytosol by going down Ran-___’s concentration gradient.
Importin
Importin
Ran-GTP
Importin
Ran-GTP’s
_________ binds Ran-___ and then binds to its cargo protein; this three-protein complex diffuse out of the nucleus by moving down Ran-____’s concentration gradient, enabling both exportin and cargo protein to move against their concentration gradients. Once in the cytosol, Ran-___ facilitates the hydrolysis of GTP to GDP and everyone loses affinity for each other; exportin and Ran-GDP then move down their respective concentration gradients unbound to one another
Exportin
Ran-GTP
Ran-GAP
In the beginning of polymerization, the shift from G-actin to __-actin is slow – called the “lag phase” or “nucleation” – but once a certain level of polymerization has occurred, a growth phase occurs in which the microfilament exponentially elongates until it eventually reachs an equilibrium phase, where the rate at which actin monomers are removed is the same at the rate at which they are added.
F-actin for “filamentous”
Polymerizing G-actin is bound to ____. Some time after polymerization, ____ is hydrolyzed. Therefore, fast polymerization results in a ____ cap and slow polymerization results in a ___ cap.
ATP
ATP
Fast = ATP cap
Slow = ADP cap
Be familiar with microfilament assemblies
Actin-polymerization-dependent ___________ and firm attachment of lamellipodium at the leading edge of the cell move the cell forward and stretch the actin ______. _________ at the rear of the cell propels the body of the cell forward, thereby relaxing tension. New focal contacts are made at the front, and old ones are disassembled at the back.
Protrusion
Cortex
Contraction
Be familiar with this picture.
Rac dominates ______________ and protrusion while Rho dominates __________________ contraction
Polymerization
Actin-myosin contraction
Nucleation of microtubules occurs by the ______-tublin complex, which is composed of 7 copies of tubulin. The 14th tubulin sites atop the 1st tubulin, leading to an assembly of 13 protofilaments.
Gamma
The kinesin cycle is a ______ ___ _____ model of movement. ATP hydrolysis in the ____________ head is accompanied by ADP release/ATP binding by the ____________ head. Conformational change in ____ ______ causes a pivot, which moves the lagging head in front of the leading head. This is called the _______________ ____________, equivalent to the length of one tubulin dimer
Foot over foot
Lagging head
Leading head
Neck linker
Power stroke
The dynein cycle is a ___________-based model. Dynein consists of a _______ that binds microtubules, a planar ring head that contains the ________ domain, and a tail that binds other microtubules or _______. ATP hydrolysis allows the ________ to attach to microtubules, and the release of ADP and Pi leads to ____________ motion in the head and stalk, the ____________ ____________. Binding of a new ATP molecule causes the __________ to release the microtubule. ATP hydrolysis leads to reversal of the rotational motion and allows stalk to reattach to microtubules
Rotational-based model
Stalk
Motor domain
Cargo
Stalk
Rotational
Power stroke
Stalk
In the myosin cycle, ATP binds to the ______ domain. When ATP is hydrolyzed, this domain changes conformation, resulting in the ___________ extending and the angle between the _______ and tail increasing. Release of Pi by the _______ domain results in initial weak contact with actin. This initial attachment is immediately followed by a much stronger attachment as the motor domain releases _________, which induces a conformational change in the motor protein, reduces the angle between the head and tail, and thereby forms the _________ ________
Motor domain
Head extending
Angle between head and tail increases
Motor domain
ADP
Power stroke
Type ___ myosin is the major motor protein in skeletal muscle
Type II myosin
When activated, myosin motor proteins pull the sarcomere together, shortening the sarcomere, but not the length of the filaments, and pulling the ___ ______s closer together
Z lines
Be familiar with this figure
Be familiar with smooth muscle cells
In smooth muscle cells, Ca2+ is released upon stimulation. This calcium binds to ____________, which undergoes a conformational change and binds ____ _____ _____ ______. Now activated, it can phosphorylate the ____ _____ _____, allowing for myosin chains to form thick filaments and “contract” smooth muscle cells
Calmodulin
Myosin light chain kinase
Myosin light chain
There are two forms of ER protein import: ___ __________ and ____ _________
Co-translation ER import
Post-translation ER import
Be familiar with the mechanism of the SRP receptor
PI(4)P is enriched at ER exit sites, the Golgi, and Golgi vesicles
PI(4,5)P2 is enriched at sites of ___________
PI(3)P is enriched in _________ _________
PI(3,5)P2 is enriched in _______ _________
PI(3,4,5)P3 is important in __________
Endocytosis
Early endosomes
Late endosomes
Phagocytosis
Regions of curved membrane that can be positive or negative curvature. Positive curvature is _______. Negative curvature is ______. These terms are used in relation to the cytosol
Convex
Concave
What are the 5 steps in generation of membrane curvature?
- Modification of phospholipid composition
- Insertion of amphipathic a-helices
- Scaffolding by peripheral membrane proteins
- Cytoskeletal changes
- Insertion of integral membrane proteins
The best studied fission of vesicles is through ________. In _______-coated vesicles, it self-assembles into stacked helical rings around the vesicle neck. It functions as a _______________ enzyme, stimulating GTP hydrolysis that causes a conformational change that ultimately leads to neck constriction
Dynamin
Clathrin-coated
Mechanochemical enzyme
Rabs regulate vesicle targeting and thethering by binding to ____________ ________
Tethering proteins
Vesicles may navigate ffrom one Rab bindg site to another down the length of a _________. Rab binding may induce conformational changes in the ______, causing the vesicle to move closer to the membrane. Rabs may also allow for multi-subunit tethering complexes to form
Golgin
Golgin
There are three current models for SNARE disassembly: distributive, __________, and ___________
Distributive
Processive
Global
What are the functions of the Golgi?
There are two models for intra-Golgi network: the _________ ____________ and the ___________ _________ models
Cisternal maturation model
Vesicle transport model
What are the three main post-Golgi trafficking pathways?
Be familiar with the coats the are responsible for movement between organelles
ERGIC
Movement of COPII and COPI vesicles
Retrieval of ER proteins
Golgi function
Most Golgi cisternae are involved in oligosaccharide processing, specifically N-linked glycosylation
Addition of mannose-6-phosphate
Trafficking of lysosomal enzymes
Know this damn figure
The G-protein _____ is ultimately responsible for the formation of SNAREpins that __________ neurosecretory vesicles. It binds its effector protein RIM1 (Rab-interacting modulator 1) and activates its binding partner Munc13-1
Rab3
Prime
Neurotransmitter release is a regulated secretory pathway
Endocytic pathways
_____________ activates Rab5 on early endosomes. Active Rab5 recruits other proteins including _______, which recruits more ______, promoting a positive feedback loop
Rabex5
Rabaptin
Rabex5
Cargo is sorted in early endosomes (sorting endosomes). Cargo to be __________ is preferentially trafficked from early endosomes, to late endosomes, and then to lysosomes. Some cargo is sorted into tubulations (tubular projections of endosome) and __________ back to the plasma membrane
Degraded
Recycled
Late endosomes mature to become lysosomes as the compartment becomes more _______, and the cell deactivates Rabs that defined the endosomes
Acidic
________________ uses recycling endosomes
Transcytosis
LDL degradation
MVBs are delivered to __________-
Lysosomes
