Molecular Biology of the Cell Flashcards
How can the formation of the nucleus be explained as an extension of the hydrogen hypothesis (formation of mitochondria through Archaeal cell engulfing a symbiotic α-Proteobacterium)?
After the α-Proteobacterium was engulfed, the nucleus formed in order to protect the host genome by either:
- Partitioning translation from transcription, so α-Proteobacterium introns which recombine into host genes can be removed.
- Preventing reactive oxygen series produced by the mitochondria from attacking host DNA.
Endosymbiosis is usually understood to be the result of an incomplete phagocytosis, how does the alternative ‘inside-out’ hypothesis explain this instead? How does this model explain nucleus formation?
- Instead of phagocytosis, the mitochondrion was engulfed via cell protrusions -blebs- which grew out around the bacterial cell wall.
- The base of these protrusions share homology with the nuclear pore complex, suggesting the original cell formed the nucleus whilst the protrusions became the rest of the cell.
Why are Asgard archaea a compelling extant model for the formation of eukaryotic cells?
Obligate syntrophy- impossible to culture Asgard archaea without another archaeon and a bacterium present.
How does the Martin and Lane hypothesis explain large genome size in eukaryotes?
- Endosymbiosis of mitochondria results in increase in bioenergetic membranes in parallel with mitochondrial genome reduction.
- Greater ATP availability permits massive increase in the number of genes that can be expressed allows innovation of new protein folds
How does DNA organisation differ between Domains?
- Bacteria- Circular, no histones, stable maintenance of chromosomes (SMC) proteins maintain ‘bottlebrush’ shaped nucleoid.
- Archaea- histones and SMC proteins
- Eukaryotes- histones and SMC proteins
How are Chromosomes organized in Interphase?
- Chromosome territories (CTs) occupy distinct but variable nuclear positions
- The interchromatin compartment (IC) contains non-chromatin domains with factors for transcription, splicing, DNA replication and repair.
- The transcriptional status of genes correlates with gene positioning in CTs- genes near centre of nucleus are more highly expressed.
How does Chromosome conformation capture help reveal the 3D organization of Chromosomes?
- DNA is fragmented using restriction enzymes.
2.DNA closely associated fragments are ligated, producing small loops.
- Loops are sequenced, and compared to a reference genome.
- Loops containing sequences from two distant points of the chromosome indicate those regions are topologically associated.
What are the 4 levels of chromosome organization hierarchy?
Level 1: Chromsome territories
Level 2: Chromosome compartments of transcriptionally active and inactive chromatin regions
Level 3: Topologically and Lamina associated domains (TADs and LADs) and
Level 4: Loops.
What are the key features of stable maintenance of chromosomes (SMC) proteins?
- Hinge domain- binds two proteins together to form ring-like complex (cohesin) around chromatids , allows ring to open and close.
- ATPase domain- may act as motor to extrude DNA through cohesin ring
- Ring is large enough to allow nucleosomes to pass through it.
How do SMC proteins form loops of DNA, and how is the size of these loops determined?
- Cohesin is loaded on the chromatin by Nipbl
- ATP-dependent cohesin movement pulls out a loop
- Antiparallel (convergent) CTCF binding sites stall movement and cohesin is unloaded by Wapl (Loops are dynamic)
What function(s) do TADs and the loops which make them up perform?
TADs provide spatial control, grouping multiple promotor/enhancer/repressor complexes into co-regulatory units
Cohesins may also act as a molecular ‘comb’ to untangle supercoiling by pushing to the boundaries where topoisomerases can resolve coils
Apart helping to organize the chromosome into TADs, what other function may cohesins perform when they loop DNA?
Cohesins may also act as a molecular ‘comb’ to untangle supercoiling by pushing to the boundaries where topoisomerases can resolve coils
What did Peter Cook controversially suggest based on the observation of discrete foci of transcription within the nucleus?
- Rather than DNA remaining stationary, and polymerases moving, DNA moves and polymerases are stationary.
- Polymerases are organized into discrete factories of 8 RNA polymerases, creating a higher effective concentration of transcriptional machinery.
- Different transcription factories specialize in different subsets of genes.
What evidence is there for fixed polymerases and DNA movement?
- Following nuclease digestion, nascent mRNA, transcribed DNA sequence and the polymerase all remain associated, suggesting they are parts of a single, stationary unit.
- During transcription of a short and long gene separated by 50 Mbp but co-regulated by TNFα, TNFAIP2 (10 kbp) and SAMD4A (221 kbp) remain in contact, this association makes sense if they are both marked for transcription at a specific factory due to being coregulated.
What are the key differences between prokaryotic and eukaryotic DNA replication?
- Prokaryotes have one replication origin, whilst Eukaryotes have many.
- Prokaryotic DNA is circular, whilst Eukaryotic chromosomes are linear, requiring telomeres for replicating ends.
- Prokaryotic replication can be continuous, whilst Eukaryote chromosomes are only replicated during S phase.
What are the stages of licensing (pre-replicative complex formation) in Eukaryotic DNA replication?
- Origin recognition complex (ORC) binds to DNA origin and Cdc6 combines with it to form ORC/Cdc6 complex
- ORC/Cdc6 complex recruits 2 Cdt1/Mcm2-7 complexes, which are loaded onto a single strand of the dsDNA.
- Regulatory proteins (Cdc45, Sld3, Sld2) bind to the Mcm2-7, forming a pre-replicative complex which can function as a helicase.
- Pre-loading complex, made up of Dpb11, GINS, and Pol ε, which replicates the leading strand, is also added.
How do Protein kinases CDK and DDK control activation of DNA replication in Eukaryotes
- CDK phosphorylates Sld3 and Sld2, allowing the Pre-loading complex (Dpb11, GINS, Polε) to bind to the pre-Replicative complex.
- DDK phosphorylates Mcm2-7 complex, allowing Cdc45 to bind
After licensing, what are the stages of initiation in eukaryotic DNA replication?
- Helicase generates ssDNA , then a single strand binding protein (RPA) binds, temporarily stabilising the ssDNA.
- DNA Pol α-primase produces RNA primers before handing over to Pol ε for replication of the leading strand.
Why does Pol α have a lower fidelity than other DNA replication associated polymerases?
Pol α lacks a proofreading function.
What is the role of Ctf4 in DNA replication?
Ctf4 facilitates interactions between various proteins on the leading and lagging strands
How are adjacent Okazaki fragments ligated to form a continuous sequence?
- When Pol ε meets an existing Okazaki fragment, it continues to extend sequence, displacing a small portion of it.
- Fen1 removes small ‘flap’
- DNA ligase 1 ligates fragments
During termination of eukaryotic DNA replication, how is the replication complex removed?
Mcm7 is ubiquitylated (but not degraded) and entire complex is removed from chromatin by p97 ATPase
Why do metazoan Origin Recognition Complexes use epigenetic markers and G4 structure to determinine the origin during DNA replication, rather than specific binding sequences like those found in Yeast?
Lack of a specific binding sequence allows greater flexibility- S phase can vary in length during different developmental stages, and different regions of the genome are transcribed throughout an organism’s life. Flexibility of replication origins allows conflicts between replication and translation to be prevented.
Why is it necessary to regulate DNA replication
- Some cell types (i.e. oocytes) must remain in a non-proliferative state and not replicate DNA for long periods.
- Chromosome origins must not fire more than once per cell cycle, to ensure each region is replicated only once.
How is DNA replication prevented in quiescent (G0) cells?
- Quiescent cells do not contain key proteins needed for pre-RC formation
- DDK and CDK not active in quiescent cells
How is it ensured each DNA region is only replicated once per cell cycle?
- Factors needed for pre-RC formation are degraded during initiation.
- The formation of the replication fork also results in Cdt1 being proteolyzed: when it is bound to DNA, PCNA (associated with Pol𝛿) acts as a focal point, allowing ubiquitin ligase to proteolyze Cdt1
What is the bifunctional role of CDK in DNA replication control?
- Low CDK in G1 permits pre-RC formation
- High CDK in S activates replication, but blocks pre-RC
Why is it necessary to have a defined timing programme during DNA replication?
- Fewer copies of replication proteins needed than if all origins fire simultaneously
- Reduce demand on dNTPs (low dNTPs can lead to genomic instability)
- Timing of replication may influence chromatin modification
- Genes replicated early in S phase will have (transient) higher copy number
What are the two forms of chromatin found in eukaryotic cells?
- Euchromatin (loosely packed, genes active).
- Heterochromatin (densely packed, genes inactive).
What is a nucleosome?
- DNA (approx 150 bp) wrapped around globular domain composed of H2A, H2B H3 and H4 histone proteins.
- Stabilised by electrostatic interaction- DNA backbone is -, histones are +
The H2A, H2B, H3 and H4 histones form the core nucleosome; what role does H1 play
Phosphorylated H1 interacts with linker DNA and is involved in higher order packing, allowing for chromatin to be compacted.
How do typical textbook illustrations of higher order nucleosome packing oversimplify the reality?
Show packing as highly organised, with regular structure. Higher order packing is actually likely quite irregular and disorganized as shown in this diagram.
What three structures/mechanisms allow for epigenetic regulation of chromatin
- Variant H2A and H3 histones
- Histone PTMs
- DNA methylation
How do variant histones such as H2A.Z differ from their canonical counterparts?
Variant or non-canonical histones differ from canonical histones in that they are DNA replication-independent, meaning they are transcribed throughout the cell cycle, not just during S phase.
What functions do variant histones perform?
Variant histones are involved in conferring local properties to specific chromatin regions. These properties often facilitate transcriptional regulation and processes such as DNA repair.
The enzymes associated with histone post translational modifications fall into which three functional groups?
- “Writers”- modify specific residues in histones (Usually residues like Arginine, Serine, Lysine, Threonine due to their side chains)
- “Erasers”- remove modifications
- “Readers”- interact with the histone modification and change the local properties of chromatin
How do histone post translational modifications impact chromatin function?
- Negatively-charged modifications (acetylation, phosphorylation) may weaken histone- DNA interaction.
- Large modifications e.g. ubiquitylation may change the structure of the nucleosome
- Modifications can be ‘read’ by proteins with specific binding domains for modification, allowing enzymes etc to be recruited which alter the properties of local chromatin.
What does this histone PTM do?
Acts as a ‘memory’ of recent transcription- Compass complex methylates it upon initiation of transcription. H3K4me3 keeps genes active by recruiting a chromatin remodeller NURF, which destabilises the nucleosomes
How does the H2K26me3 histone PTM prevent the accidental initiation of further transcription within the gene body whilst a gene is being transcribed?
1.SetD2 moves with RNA pol II, methylating H3K36.
2. Histone deacetylases bind to H3K36me3, deacetylation increases the positive charge of the histones, causing them to associate more strongly with surrounding DNA.
How does DNA methylation regulate transcription?
- DNA methyltransferases (DNMTs) establish and maintain methylation of cytosine.
- methylation can then either:
i. Directly block transcription factors.
ii. Be recognized by proteins (MBDs or DNMTs) which recruit transcription inhibitors.
How are DNA methylations removed?
TET oxidises base, causing it to be read as damaged by base excision repair system, which removes and replaces it.
How does DNA methyltransferase 1 (DNMT1) ensure both strands of DNA remain fully methylated during semi-conservative replication?
DNMT1 only methylates hemimethylated DNA- where one strand is methylated and the other isn’t. This creates two fully methylated sets of dsDNA.
How are histone modifications retained after DNA replication?
- PRC2 complex binds to modification on parental nucleosome and becomes active.
- PRC2 modifies neighboring nucleosomes, spreading modification along chromatin molecule.
- Blocking domains prevent PRC2 from extending too far
How are sister chromatids held together after replicating in the pre-meiotic S phase?
Meiotic cohesin ensures they remain associated.
What two different alignment mechanisms do eukaryotes use to bring homologous regions into close proximity during recombination in meiotic prophase I?
- Synaptonemal complex: compares sequences of chromosomes and aligns a small matching region, before moving down chromosome and aligning the rest based on that initial alignment.
- Horsetail movements: telomeres are attached, then chromosomes are ‘shaken out’ aligning them. Found in a few eukaryote species, such as fission yeast (S. pome)
What is the mechanism of homologous recombination meiosis prophase I?
- Nuclease(Spo11) makes double strand break in one of the homologous chromosomes.
- 5’-3’ nuclease (MRA) resects ends leaving 3’ overhang
- 3’-strand invades the other homologous chromosome, extended by a polymerases (Rad51) forming D-loop (displacement loop)
- Homologous chromosome catches other side of initial double strand break forming
- Ends are religated to form double Holliday junction
- Holliday junction resolvases nick and religate DNA
- Depending on process of nicking and ligating product can be either crossover or non-crossover
How do chromosomes attach correctly to the meiotic spindle during meiosis I?
Cohesin maintains association of homologous chromosomes after recombination is completed.These links are called chiasmata.
In meiosis there are potentially four kinetochores, which could potentially result in incorrect attachments. Which proteins prevent this from occuring?
Monopolins- modify sister kinetochores so they behave as a single unit
In meiosis there are potentially four kinetochores, which could potentially result in incorrect attachments. Which proteins prevent this from occuring?
Monopolins- modify sister kinetochores so they behave as a single unit.
Total loss of cohesin during meiosis I would result in the sister chromatids separating, and so not being configured correctly for meiosis II. How is this prevented?
- Shugoshin (guardian spirit) protein protects cohesin in vicinity of centromere by recruiting PP2A phosphatase, which makes cohesin resistant to cleavage
- Cohesin in chromosome arms allows homologous chromosomes to separate, whilst cohesin maintained at centromere keeps sister chromatids linked.
What prevents DNA replication between meiosis I and II?
- High CDK activity is maintained throughout meiosis via the limitation of cyclin B proteolysis.
- High CDK activity prevents DNA helicases from loading onto origins of replication
Which two RNA polymerase-catalysed reactions are necessary for transcription?
Transcription encompasses two opposite reactions: nucleotide addition and excision of incorrect nucleotides.
Outline the process by which DNA is guided into an RNA polymerase, and then transcribed into RNA during the elongation stage of RNA transcription.
- DNA enters the RNA polymerase through the jaws and is guided along the bridge until it meets the wall.
- Rudder and clamps secure DNA and allow strands to be separated.
- Nucleotides enter through secondary channel and bind to ssDNA.
- RNA is then guided out of the enzyme by the rudder.
What components of RNA polymerase are likely retained from a common ancestor in Bacteria, Archaea and Eukaryotes?
Fe-S cluster, the catalytic centre of the enzyme, and accessory proteins associated with elongation
RNA polymerase initiation factors evolved independently in Bacteria, Archaea and Eukaryotes, in response to what shared problem?
Initiation factors likely evolved as a way of more tightly regulating where RNA polymerases bound to DNA.
What are the 6 subunits of bacterial RNA polymerase, and what are their functions?
Subunits: α, α, β, β’, σ, ω
- β and β’ - responsible for catalysis
- σ (and α)- make contact with DNA
- ω and α- scaffold other subunits
How does bacterial RNA polymerase make contact with the right sites on the DNA?
- 1D Hopping- continuous association and dissociation, moving along the DNA
- 1D Diffusion- sliding along DNA
- Intersegmental transfer: movement from one DNA site to another that is nearby due to DNA looping.
- 3D diffusion- passive diffusion whilst dissociated- MOST IMPORTANT DRIVING FACTOR
How can a protection assay be used to identify RNA polymerase binding sites in DNA?
- DNA is incubated with RNA polymerase.
- Endonuclease which cuts at random sites is added
- Electrophoresis will produce bands associated with cuts at all sites, except those where the RNA polymerase is bound.
How does the σ subunit of bacterial RNA polymerase change the DNA affinity of the entire complex when it makes contact with a DNA binding site?
- Specific domains of the σ subunit bind to discrete regions in the promoter
- σ is primarily composed of alpha helices, every 3rd amino acid is a specific residue which is able make contact with the exposed nucleotides in the dsDNA binding site.
- σ subunit sigma factor contains DNA binding domain, which is usually masked, but when it makes contact with a DNA bind site, it becomes exposed, changing the affinity of the entire RNA polymerase.
How do σ factors (subunit of prokaryote RNA polymerase) help to regulate gene expression?
- Organisms possess multiple different σ subunit variants, with different promoter preferences. Allows control of where RNA polymerases bind depending on evironmental/developmental conditions.
- anti-σ factors bind to σ subunits to prevent them binding to DNA, can be dependent on environmental conditions such as nutrient availability.
What are the three forms that the bacterial RNA polymerase-DNA complex moves between during initiation?
- Closed: When DNA has only partially engaged with RNA polymerase and is still double stranded
- Intermediate: DNA bends, and σ region 1.1 makes contact, helping to bring it into the channel.
- Open: DNA enters the channel and the strands are separated (again by σ subunit), allowing first dNTP to bind.
What is’ scrunching’ during bacterial RNA polymerase initiation?
Once the DNA strands have been separated, the RNA polymerase produces short RNA oligos. When a fragment >10 nt is produced (usually after about 5 cycles0, the Sigma factor is released and elongation initiates.
How does the σ subunit of bacterial RNA polymerase separate the DNA strands during initiation of transcription?
σ subunit flips two nucleotides (adenine at -11 and thymine at -7), helping to break the two strands apart.
What are the topological consequences of transcription, and how are they resolved?
Transcription results in supercoiling either side of the transcription site. It would be problematic for the transcriptional machinery to encounter these coils, so Gyrase and topoisomerase resolve these structures.This results in heavily transcribed regions having very low levels of supercoiling/DNA-DNA interaction
How does Rho independent (intrinsic) transcription termination occur?
Terminator region of DNA containing two-fold symmetry is transcribed, producing an RNA sequence which binds to itself to form a terminator hairpin, which induces conformational change in RNAP, displacing it.
How can Rho-independent termination be prevented under certain circumstances, and why is this desirable?
In certain conditions (i.e. exposure to certain metabolites) mRNA can instead form an antiterminator hairpin, which does not induce RNAP conformational change.
This is desirable as it allows for the transcription of certain coding regions (occurring after the terminator) to be dependent on the presence of specific environmental conditions.
How does Rho-dependent termination occur?
- DNA sequence is transcribed to produce high cysteine content rho-utilisation (RUT) RNA sequence
- Rho termination complex binds to RUT and moves along RNA towards RNA polymerase. through helicase activity.
- Rho termination complex reaches RNAP and displaces it.
Plants possess two extra RNA polymerases in addition to those found in other prokaryotes, Pol IV and Pol V. What is their role and how did they evolve?
They are involved in the production of non-coding RNAs. They probably evolved from Pol II
How do basal transcription factors recruit RNA pol I during transcription initiation?
- Upstream binding factor (UBF) binds to Upstream promoter element, and then rolls up DNA so it is in proximity with the core promoter
- UBF recruits complex including TBP, which recruits Pol I
How many different types of promoter can bind RNA Pol III?
3- they consist of conserved elements arranged in alternative configurations
Outline the initiation mechanism broadly followed by all RNA polymerases during eukaryotic transcription initiation.
- Polymerase is recruited by basal transcription factors, forming a pre-initiation complex (PIC)
- Complex enters intermediate open (unstable) state
- polymerase scrunches, and is then released from the initiating complex.
How is histone organization impacted during transcription?
As first pol II moves past H2A/2B dimer is ejected, whilst H3/4 tetramer is maintained with a lower affinity for DNA. If no more pol II pass that point in rapid success, the histone will reassemble.
At high level of transcription, additional pol II will pass the lower affinity tetramer in rapid succession, resulting in the entire histone octamer being ejected.
Describe the lariat (lasso) mechanism of RNA splicing and how it ensures external energy is (theoretically) not required for the reaction.
- A lariat is formed when the intron is cleaved at the 5′ splice site and the 5′ end is joined to a 2′ position at an A in the branch point sequence.
- The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together.
- Number of phosphodiester bonds is maintained during the reaction, meaning the process is not reliant on external energy.
Describe the composition of the U2 spliceosome
U2 spliceosome is composed of:
- 5 snRNAs.
- 41 proteins which associate directly with snRNA to form snRPs.
- 70 proteins necessary for assembly of the spliceosome (bind to RNA to construct RNA-based centre for transesterification reactions).
- ~30 proteins which recruit factors involved in processes such as regulation and quality surveillance.
These are organized into 5 small nuclear ribonucleoprotein (snRP) complexes.
U1 is the first snRP to contact pre-mRNA during U2 intron splicing. Explain how U1 recognises its target.
The snRNP U1 recognises the 5’ splice site (left) AG of the pre-mRNA by base pairing of its SnRNA.
Describe the assembly of the U1 and U2 complexes during U2 spliceosome formation
- The first snRP, U1, recognises the 5’ splice site (left) AG of the pre-mRNA by base pairing of its snRNA.
- U2 binds to the branch point with assistance of 3 auxiliary factors: U2 auxiliary factors U2AF65 and U2AF35 (bind at Polypyramidine tract and 3’ splice site respectively) and branch point binding protein (BBP).
- U2 displaces adenine at branch site, making it protrude from intron and so more susceptible to nucleophilic attack.
After U1 and U2 have bound to intron, how is the rest of the U2 spliceosome assembled, and how does it become catalytically active?
- Super complex composed of snRNPs U4/U5/U6 is incorporated. U4 and U6 snRNA interact by base pairing, while the U5 snRNP associates by means of protein-protein interactions.
- Completed spliceosome undergoes rearrangement: U1 and U4 are ejected, causing U6 to pair with U2 via snRNA interaction, forming the catalytic spliceosome.
How do transesterification reactions form the lariat during U2 splicing?
Introns are highly variable in length. What two mechanisms involving SR proteins (composed of RNA Recognition Motif and Arg-Ser rich domain) are used to define intron size within an mRNA molecule?
Two similar mechanism in which SR proteins form a chain which is associated with RNA:
- Intron definition: SR associated RNA region is defined as intron
- Exon definition: regions flanking SR associated RNA are defined as introns
In organisms with large introns, such as mammals, backsplicing can occur. How is exon backsplicing (in which two ends of an exon are accidentally ligated, forming a ring) typically avoided during splicing?
Steric encomberance: RNA molecule is large and complex enough (and in the case of exon-definition associated with SR proteins) to prevent complex from assembling correctly if two ends of an exon are loaded, forcing the complex to rearrange.
How can backsplicing still sometimes occur, and why can it be beneficial?
- Very long (or multiple connected) exons can form a large enough loop to accomodate the spliceosome, making steric encumbrance no longer an issue.
- Backspacing generates circRNAs, which are exonuclease resistant and can perform a range of cellular roles by interaction with RNA and proteins, for example, serving as sponges which ‘soak up’ miRNAs.
How does miRNA processing differ between plants and animals?
- In animals Drosha removes tail to form Pre-miRNA and Dicer removes loop structure to form miRNA duplex. In plants, DCL1 removes tail to form Pre-miRNA and removes loop structure to form miRNA duplex- one enzyme performs two modifications.
- In animals, nuclear export occurs before loop is removed, whereas in plants it occurs after
- In animals, mature miRNA ends in -OH group, whilst in plants it ends in -OCH3 methyl group
What is the role of miRNA in establishing the meta/proto-xylem gradient in plants?
- SHORT ROOT (SHR) transcription factor is made in central vascular tissue, it then moves into the endodermis where it enters the nucleus.
- SHR-transcription factor activates a gene encoding a miRNA called 165/6 in endodermal nuclei.
- miR165/6 moves into adjacent cells, forming a ‘morphogen’ gradient (high abundance in dark green cells, low in light green cells).
4.An mRNA encoding the PHABULOSA (PHB) transcription factor is down-regulated by miR165/6 in a dose-dependent manner
- High levels of PHB specify metaxylem, low levels specify protoxylem.
This produces a Meta/Proto- xylem gradient
What role does siRNA gene silencing play in developing an immune response towards viruses?
- Sometimes, viral ssRNAs are improperly capped, RDR binds and makes the RNA double stranded.
- Dicer detects this dsRNA and cleaves it into siRNAs
- siRNAs can then act as primers, binding to fully capped viral ssRNAs, amplifying gene silencing.
Describe the overall structure of a nuclear pore complex.
Large- 500-1000 proteins, assembled from multiple copies of ~30 nucleoporins.
Two main features:
- Symmetric core:
- 3 rings
- central pore
- Asymmetric nuclear and cytoplasmic peripheries:
-Nuclear basket
-Cytoplasmic filaments
What is the the key difference between nuclear export and import by Exportin and Importin respectively?
Key difference is type of binding between Importin/Exportin and RanGTP
What is the the key difference between nuclear export and import by Exportin and Importin respectively?
Key difference is type of binding between Importin/Exportin and RanGTP
Describe mRNP complex formation
- 5’ methyl guanosine cap binds (Cap-bind proteins) CBP complex; CBPs promote splicing
- After splicing Exon junction complex (EJC) and Serine-Arginine (SR) proteins are deposited in the coding region. First EJC and CBC together recruit TREX and Dbp5 DEAD-box RNA helicase.
- Exon interactome (green) helps folding and packaging the coding region, including ATP-dependent DEAD-box proteins.
- After 3’ UTR cleavage and polyA synthesis, PolyA tail binding Protein complex ( PABPC) forms
After the formation of the mRNP complex, how is mRNA exported from the nucleus via bulk mRNA transport?
- Diffusion of mRNP from the site of transcription to NPC is a rate limiting step (seconds up to minutes).
- TREX and SR proteins recruit NXF1-NXT1 receptor ( Nuclear RNA export factor 1/ NTF2-like export factor 1). Interaction with TREX induces the affinity of NXF1 to RNA binding.
- Docking of mRNP-receptor complex to the nuclear basket
4.Translocation is very quick (~20 ms)
Describe nuclear envelope budding as an alternative form of mRNA export
- RNP granules can exit nucleus by budding
- Local lamina disassembly allows membrane coat molecules to distort inner nuclear
membrane and induce docking and budding
3.Vesicles in perinuclear space fuse to outer nuclear membrane or ER membrane
Describe how pulse-chase analysis can be used to determine how proteins move through a secretory pathway.
- Radioactively labelled amino acids are added to the tissue- these will be incorporated into proteins- ‘pulse’.
- Add excess of unlabelled amino acids- ‘chase’.
- As radioactive proteins move through cells, the proteins that take their place will be unlabelled due to chase.
- Prepare tissue for electron microscopy at various times.
In each microscopy image the radioactive proteins can be identified, allowing their progress to be tracked between images
What is a polyribosome? How might these inform the folded structure of the the endoplasmic reticulum?
- Group of ribosomes all translating the same mRNA
- Folds of ER might serve to increase its surface area to allow polyribosome formation.
Describe the structure of a signal peptide
- Polar, hydrophilic ends
- ~20 amino acid hydrophobic region.
- Hydrophilic/Hydrophobic regions allow peptide to span membrane
Describe how the Signal Recognition Particle targets proteins to the ER membrane.
- ER proteins have a hydrophobic signal peptide of ~20 residues at the N terminus
- The Signal Recognition Particle (SRP) binds the signal peptide as it emerges from the ribosome and arrests translation
- The SRP docks the ribosome onto protein translocator in the ER membrane, this opens the pore in the translator.
- SRP then leaves and translation reinitiates so translocation is co-translational
Describe the features of the signal recognition particle and their functions.
- SRP-54: Recognises signal peptide:
-features methionine bristles
- SRP54 binds ~20aa hydrophobic sequence on signal peptide
- Affinity for SP is relatively low (sub-nM KD)
- SRP-9 and SRP-14: Arrest translation
- Arrests translation via competition with elongation factor binding on the ribosome
- SRP-68 and SRP-72: Dock with ER
What is the translocon and what is its role in transport of proteins across cellular membranes?
- Universally conserved protein-conducting channel, referred to as the Sec61 channel in eukaryotes and as the SecY channel in prokaryotes.
- Single copy of the heterotrimeric Sec61 or SecY complex forms the channel, resulting in a narrow pore
- Positively charged region of signal peptide interacts with negatively charged residues on cytosolic side of pore
- signal sequence is intercalated into the lateral gate and the following segment passes through the actual pore as a loop. This results in displacement of the plug domain of the channel.
How are transmembrane domains defined by the translocon (translocation channel)?
How are transmembrane domains incorporated into the membrane by the translocon
Lateral gate opens in the translocon, allowing the TMD to move out into the cytoplasm where it is held via hydrophobic interaction.
Beginning in the 1980s, there was a debate as to how proteins exited the endoplasmic reticulum, the two main hypotheses were Bulk Flow and Signal Mediated Export. What were the different assumptions and predictions of these hypotheses?
How did the debate surrounding the Signal Export Mediated and Bulk Flow hypotheses become more nuanced over time?
Both now accept that export is the default.
Describe how the QC glycocode selectively retains misfolded proteins within the ER/
- Oligosaccharide transferase N-links a high mannose glycan to asparagine residue in the nascent polypeptide
- The two terminal glucoses are removed by Glucosidase I and Glucosidase II,
- Calnexin (CNX) or Calreticulin ((CRT) soluble lumenal form of CNX) bind to remaining glucose (Glu1)
- Glu1 is removed as the protein folds
- The rate of dissociation from CNX or CRT governs the export rate
- UDP-glucose:glycoprotein glucosyltransferase 1 (UGT) selectively adds glucose to unfolded proteins.
- How does it discriminate?
a. exposure of hydrophobic sequences
b. accessibility of the glycan core - Adding of glucose to unfolded causes proteins to continually re-associate with CNX and CRT and be retained
Some proteins retained in the ER by the QC glycocode cannot be folded, and are fed into the ERAD pathway to be degraded. Describe the ERAD pathway.
- ER mannosidase I converts unfolded proteins to Man-8
- Reduces interaction with CRT/CNX and UGT
- If the protein is not folded – Man8 glycans become substrates for ER mannosidase-II
- EDEM = ER Degradation Enhancing Mannosidases
- Man-7 associates with specific ‘ERAD’ lectins
- The unfolded protein is fed back through the Sec61 pore
- It becomes ubiquitinated and targeted to the 26S proteasome
The observations regarding cargo molecule concentration by Bill Balch are, in fact, the result of an active mechanism, the COPII Sec24p receptor system, making them incompatible with the bulk flow hypothesis. Describe the COPII Sec24p receptor system.
- Sec12p activates Sar1p by exchanging GDP for GTP
- Sec12p is resident in the ER and ensures Sar1p is only activated there
- Sar1p-GTP embeds in the membrane
- The Sec24p/Sec23p component is recruited to the membrane by Sar1p-GTP
- Membrane cargo protein bind via their cytoplasmic tails
- Sec13p/Sec31p complex binds to Sec24p/Sec23p to form the coat
What are extracellular vesicles?
- Intralumenal vesicles formed in the late endosome and expelled through fusion of MVBs with the PM
- Non-replicating structures delimited by a lipid bilayer
- Contain cargoes specific to producing cell (RNA, proteins, lipids, metabolites)
- Membrane proteins retain their plasma membrane topology- the face the same direction as they do on the PM, rather than being inverted like in the early endosome.
- All living cells across all kingdoms produce and release EVs into the extracellular milieu
How did Ernst Ruska’s work with Transmission Electron Microscopy in the 1960s help to build the case for the existence of Extracellular vesicles?
- Exosomes were directly observed for the first time, and appeared in Micrographs from many organisms.
- At this stage they were assumed to be inert cellular debris or even artifacts of electron microscopy itself
How did Rose Johnstone and Philip Stahl’s research into reticulocytes (immature red blood cells) in the 1980s accidentally demonstrate that the release of extracellular vesicles was a regulated process?
- Reticulocytes contain Transferrin Receptors (TfR) at their PM that bind di-ferric-Transferrins (Tf) via their Extracellular Domain. These receptors are lost during maturation.
- Both were interested in visualizing the path taken by TfR-Johnstone used fluorescent and radio-labelled TfR, whilst Stahl used radio-labelled Tf
- Found that prior to maturation, TfR/Tf complex dissociated in the early endosome and components were recycled.
- Determined that, during maturation, TfR was instead expelled from the cell via an exosome- this demonstrated the release of these vesicles was a regulated process.
How did Graça Raposo’s research into the Major Histocompatibility Complex Class II prove that extracellular vesicles were biologically active, rather than inert?
- Demonstrated that EVs could possess MHC Class II proteins on their membranes,
- Demonstrated MHC Class II proteins were functional in EVs by incubating them with T cells and showing that they stimulated an immune response.
What function of extracellular vesicles was demonstrated by papers such as Valadi et al. (2007)?
- EVs contain sRNAs and mRNAs, many of which are specifically packaged exclusively into exosomes
- Cells can take up exosomal RNAs produced by other cells- suggests role in cell-to-cell communication.
How are RAB5-GTPase (small GTPase) labels added to multivesicular bodies in order to prepare them for transport?
How are the RAB5-GTPase labels on multivesicular bodies utilized during their transport?
- RAB5-GTPase binds to kinesin, tethering the multivesicular body to microtubule.
- Different RABs specify destination, e.g. RAB27b directs towards plasma membrane.
