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BIOL 200 > Nucleo-Cytoplasmic Transport + Translation > Flashcards

Nucleo-Cytoplasmic Transport + Translation Flashcards

1
Q

What are the sites of nucleocytoplasmic transport?

A

Nuclear pore complex:
- 30x bigger than ribosome (huge!) /125 megadaltons
- ~50 (yeasts), ~100 (vertebrate) different proteins
- On nucleoplasm side → baskets
- On cytoplasmic side → filaments

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2
Q

On what does transport through NPC depend?

A

Depends on reversible hydrophobic intractions
(smaller molecules can pass on their own, bigger need to be transported)

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3
Q

What are FG nucleoporins?

A

Specific class of nucleoporins (which are the proteins that make the structural component of NPCs)

  • Characterized by the presence of phenylalanine-glycine (FG) repeat sequences
  • FG repeats form intrinsically disordered domain
  • Hydrophobic

Proteins make their way through the pores by interacting with FG repeats

*in pores: Hydrophilic region decorated with FG repeats

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4
Q

Why is nuclear transport important for nuclear proteins?

A

All nuclear proteins are synthesized in the cytoplasm and import through NPCs (Nuclear Pore Complex)

These proteins contain nuclear localization signal (NLS) → Specific sequence of amino acids + protein that directs the protein to be transported to the nucleus

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5
Q

Which expriment proved that nuclear localization signal (NLS) is required for targetting the nucleus?

A

pyruvate kinase found in cytoplasm
NLS-pyruvate kinase found in the nucleus by fluorescence

Identified with SV40, using T-antigens that enter the nucleus during infection. Mutations in T-antigen didn’t allow virus to infect nucleus as well

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6
Q

Which proteins are required for nuclear import? What do they interact with?

A

Ran (Ras-related nuclear protein): monomeric G protein that exists in 2 conformations → one bound to GTP and one bound to GDP

Nuclear Transport Receptors (on family is Importins): proteins bind to NLS domains present on cargo proteins to facilitate transport through pore by associating with FG repeats on the nucleoporins

*They interact with NLS

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7
Q

What enzyme is reponsible for conformation changes in G-protein?

A

Active form = bound to GTP
Inactive form = bound to GDP

On → GAPs → Off
Off → GEFs → On

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8
Q

Explain the mechanism of nuclear import of NLS-containing cargo proteins.

A

*Driven by concentration gradients
1. Importins recognize NLS on cargo and forms complex with it.
2. By import complex concentration gradient + importin’s interaction with FG repeats, moves into NPC
3. Ran GDP + GEF → Ran GTP
4. Ran-GTP in nucleoplasm has higher affinity than cargo for importins → Ran-GTP-importin complex (cargo freed)
5. Ran-GTP-Importin complex moves back to cytoplasm by concentration gradient
6. In cytoplasm, GAP hydrolyzes Ran-GTP → Ran-GDP → confirmational change → releases importin → importin is now free to bind to a new Cargo protein
7. When Ran-GDP becomes to concentrated on cytoplasmic side, makes its way back to nuclear side, where GEF converts it into Ran-GTP
Continues as a loop!

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9
Q

On which side of the NPCs are found GEF and GAP?

A

GEF found in nucleoplasm: Ran-GDP → Ran-GTP, maintains high concentration of Ran-GTP so it can bind to importin

GAP found in cytoplasm: Ran-GTP → Ran-GDP, to dissociate from importin

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10
Q

What is the role of Exportin t?

A

Exports tRNAs:
1. Binds fully processed tRNAs (processed entirely in the nucleus) + Ran-GTP → passes through NPCs to go on cytoplasmic side as a trimeric complex

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11
Q

Which RNA are exported through association with Ran?

A
  • t RNAs (with Exportin t + Ran)
  • Ribosomal subunits
  • Some specific mRNAs that associate with specific hnRNP proteins (ex: HIV Rev)

**Most mRNA = Ran-independent process using an mRNA exporter

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12
Q

What does hnRNP stand for? What is their role?

A

Heterogenous nuclear ribonucleuprotein
Family of RNA binding proteins involved in RNA splicing, transport, metabolism, etc.
Mostly associated with pre-mRNA in the nucleus.
They bind to RNA by RRM or another RNA recognition domain

*SR proteins (Serine-Arginine rich proteins bind RNA exons, regulation of alternative splicing)

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13
Q

Explain a model of mRNP export through the NPCs?
(The simple one)

A

*Ran-independent
*Transport of mRNA from nucleoplasm to cytoplasm for protein synthesis (ribosomes are in the cytoplasm)
- NXF1-NXT1 heterodimer binds to mature RNA (they are not hnRNPs) → NXF1 =RNA binding proteins → recognizes cap and poly(A) tail associated with mRNA
- Together they bind mRNA cooperatively with specific mRNP proteins (including SR proteins) → froms mRNP complex
- Form a domain that interacts with FG repeats in nucleoporins

*ATP dependent process as Dpb5 undergoes phosphorylation as bound to filament of the NPC

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14
Q

What is mRNP?

A

mRNP= messengerRNA ribonuclear protein
It is a complex formed of mature RNA (spliced, capped, poly(A)) + proteins bound to it
These proteins can be NXF1-NXT1 or other proteins with RNA binding domains
This complex is essential:
- transport of mRNA across nuclear membrane
- processing
- translation

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15
Q

What are Balbiani rings?

A

Found in Chironomous tentans (an insect)
Balbiani rings = structure of mRNP capable of going through nuclear pore.
1. hnRNPs bind RNA
2. As its being transcribed, packaged into Balbiani rings for transport

They are insect polytene chromosomes (in salivary glands for sticky saliva) that provide a system where transcription and mRNP export can be miscroscopically imaged

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16
Q

Explain an mRNP export model involving Balbiani rings

A
  1. mRNP forms balbiani rings (with all mRNP components) → found in insects
  2. Balbiani rings enter the nucleoplasm NPC baskets
  3. 5’ end of mRNA part of the Balbiani ring goes through the pore first and immediatly is bound by ribosomes that start translation
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17
Q

Can pre-mRNA leave the nucleus through NPCs?

A

No, it stays in nucleus until full maturation (not yet well understood how)

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18
Q

Does the same set of protein stay associated with mRNA on the nuclear and cytoplasmic side of NPCs?
What happens?

A

No
1. Proteins associated mRNA on nucleoplasmic side are removed by RNA helicase ATP-dependent process direclty when entering cytoplasm
2. Cytoplasmic proteins bind to mRNA
3. Nuclear proteins are sent back to nucleoplasm through NPC

Callled Cytoplasmic remodelling
ex: PABPN1 and PABPN2 replaced by PABPC1 and PABPC2

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19
Q

What part of the NPC is responsible for removing nuclear protein attached to RNA when transported from the nuclear to the cytoplasmic side?

A

Filaments of the NPC on the cytoplasmic side
- Through RNA helicase activity

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20
Q

What are the 3 general roles RNA in protein synthesis?

A
  • mRNA = information for the amino acid sequence
  • tRNA = required to bring amino acids into the ribosome, helps read codons on mRNA
  • rRNA = Assembling giant protein synthesis machinery
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21
Q

What % of the cell’s RNA is rRNA? How is it arranged?

A

rRNA = ~80% of total cellular RNA
Arragend in repetitive clusters

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22
Q

What is the structure of a Bacterial Ribosome?

A

Large subunit = 23S rRNA (synthesized by RNA pol II) + 5S rRNAs (synthesized by RNA pol III) + 31 proteins = 50S
Small subunit = 16S rRNA (synthesized by RNA pol II) + 21 protein = 30S

Assembled ribosome = 70S

23
Q

What is the structure of a Eukaryotic Ribosome?

A

Large subunit = 28S rRNA (synthesized by RNA pol II) base pairing with 5.8S (synthesized by RNA pol III) + 5S rRNAs (synthesized by RNA pol III) + 50 proteins = 60S
Small subunit = 18S rRNA (synthesized by RNA pol II) + 33 protein = 40S

Assembled ribosome = 80S

24
Q

Which shows greater variation through species in rRNA, 2ndary structure or nucleotide sequence?

A

Secondary structure of rRNA = highly conserved → stem-loop structures
*Similar in bacterial and eukaryotic

25
Q

What are 2 important structures of tRNA for translation?

A

3’-CCA is where tRNA will attach to its specific aminoacyl-tRNA synthetase (enzyme) which will ensure that the appropriate amino acid binds to the tRNA

Anti-codon loop at the bottom (specific to the amino acid that will bind to tRNA)

26
Q

How do amino acids bind to tRNA?

A
  1. Aminoacyl tRNA synthetases recognizes and bind to their specific cognate tRNA
  2. Linkage of Amino Acid to its corresponding tRNA through ATP phophorylation → AMP + PPi
    Formation a high energy ester bond at the C-terminus of the amino acid
  3. Anti-codon of tRNA binds to codon (is selected)
27
Q

What features that make the code of mRNA considered “degenerate”

A
  1. Aminoacyl-tRNA Synthetases recognize and bind to their cognate tRNAs
  2. A single acminoacyl tRNA synthetase can bind to more than one unique tRNA → allows multiple anti-codons to be associated with the same amino acid
  3. A single tRNA can bind to more than one single codon → also allows different an amino acid to be associated with multiple different codons

*When ribosomes read codons, they create a special environment so that the 3’ or 5’ nucleotide of the codon can sometimes interact in a non-Watson-Crick manner

28
Q

What are examples of a “degenerate” mRNA code in the ribosome?

A
  • 6 different codons can code for Leucine
  • 6 different codons can code for Serine
  • 6 different codons can code for Arginine
29
Q

Which tRNA is absolutely needed to start synthesis of a polypeptide chain?

A

tRNA Met initiator

*In bacteria, modified by addition of a formyl group (CHO) not added in normal tRNA Met
In Eukaryotes and Acheaeans, stays the same

  • tRNAi Met = exclusively for initiation of a polypeptide chain, there is another tRNA Met for later addition of methionine
  • tRNAi Meth Recognizes the P site on ribosome
30
Q

What are the steps leading to the assembly of the pre-initiation complex?

A
  1. eIF2 ternary complex formation: tRNAi bound with Meth binds with eIF2-GTP (eukaryotic Initiation Factor 2 = G-protein)
  2. Complex + small subunit of the ribosome + eIF5 (which binds to eIF3 and eIF1) → 43S complex formation → 43S preinitiation complex
31
Q

How can protein synthesis be negatively regulated at initiation of translation?

A

By phosphorylation of eIF2 → eIF2-GDP instead of eIF2-GTP

32
Q

How is the state of the small subunit leading to formation of the pre-initiation complex or translation?

A
  • Has E, P, A sites
  • eIF3 and eIF1 are bound to E site
  • eIF1A bound to the A site
  • P site is available to receive tRNAi Meth w/ 2-GTP
33
Q

What structure of mRNA is required for efficient translational initiation of eukaryotic mRNAs?

A

The 5’ cap, which only class II mRNA have, dimeric guanylyltransferase binds to phosphorylated CTD of RNA pol II

That’s why only class II mRNA are efficiently translated and lead to protein synthesis

Cap-binding protein (binds 7mGDP cap) required for efficient translational initiation

34
Q

What is the role of eIF4 in the preinitiation complex?
Specific roles of eIF4E and G

A

Role = mRNA activation ATP-dependent
eIF4 = multi-subunit protein complex → crucial role in recruiting an mRNA (4E, 4G, 4A)
- eIF4 E is the cap binding protein, binds to 5’ cap of the mRNA + helps positionning of other subunits
(Over expression of eIF4 associated with tumour formation because too much translation, very tightly regulated levels in cells)
- eIF4 G = recruits small subunit of ribosome → binds eIF3 (on P site) and PABPC (associates with poly (A) tail, cytoplasmic version) with its 2 subunits 4A and 4B
*4B gets in later at activation of mRNA, not part of the eIF4 complex

35
Q

What is the importance of the interaction between eIF4G and PABPC?
What does it favour?

A
  • PABPC is bound to poly (A) tail (3’-end)
  • eIF4G is bound at 5’ end of mRNA
    By binding to it, mRNA forms a loop
    This loop favours reinitiation of translation for formation of multiple proteins
  • Helps with mRNA stabilisation
36
Q

Where is the start codon compared tha eIF4 complex on the mRNA?

A

eIF4G bound at 5’-end, AUG is downstream from it (not directly beside)

37
Q

What are the roles of eIF4A and eIF4B in the preinitiation complex?

A

eIF4A-RNA helicase (ATP) → Uses ATP to unwind mRNA 2nd structures in the 5’ region + motor carrying other eIF4 proteins along mRNA as translation goes → Scans until the complex reaches to the initiation codon (AUG)

eIF4B enhances eIF4A activity, acts as a co-factor

(eIF4E would not stay at the 5’ end bound to the cap, but would follow the complex through formation of a second loop)

38
Q

What happens when the scanning complex of translation reached the AUG codon?

A

Interaction at P site between anti-codon loop on tRNAi Meth and AUG codon → conformational changes → GAP eIF5 hydrolyzes eIF2-GTP to eIF2-GDP → conformational change → becomes the 48S initiation complex → All initiation factors will dissociate:
- eIF1, eIF3, eIF5 → frees the E site
- eIF2-GDP
- eIF4 with all subunits (4E, 4G, 4A, 4B)

Then, large subunit joins → 80S Initiation Complex (E and A sites are empty, chain elongation factors join, tRNAi Met is in the P site)

39
Q

What are the 2 parallel events happening during pre-initiation of translation?

A
  1. activation of mRNA through binding ot eIF4 complex
  2. Formation of the 43S complex on small ribosomal subunit (40S) = Small subunit + tRNAi Meth + 2-GTP + eIF5 binding to eIF1 and eIF3 that are on E site
39
Q

What is the effect of a longer poly (A) tail on mRNA vs a shorter one?

A
  • More stable to exonuclease degradation
  • more PABP protein can bind to it (3’-end) → more interaction with eIF4G (5’-end) → stronger mRNA loop → better reinitiation of translation
40
Q

Approximately how long does the scanning complex go before reaching AUG?

A

about 100 nucleotides

41
Q

Explain the elongation step of translation.

A
  1. EF1-alpha-GTP bound to tRNA enters the A site
  2. Try interaction between codon and their anti-codon at A site
  3. Keep trying until base-pairing between anti-codon and codon → binding of complex to A site → EF1-alpha-GTP hydrolysed to EF1-alpha-GDP → EF1-alpha falls off + conformational change in whole ribosome
  4. Conformational change → bring amino acids linked to tRNA of P and A sites very close →
  5. Peptidyltransferase activity (formation of peptide bond)
42
Q

What is a ribozyme?

A

A ribozyme is an enzymatically active RNA molecule (not protein)
- The ribosome contains one that catalyses peptidyltransferase activity (23S in bacteria and 28S in eulkaryotes)

43
Q

What is the important of the ribozyme in Peptidyltransferase?

A
  • Occurs when amino acids linked to tRNA on A and P sites come to close proximity
  • rRNA mediated (Ribozyme), folds up into 2nd structure
  • Ribozyme: in bacteria = 23S rRNA, in Eukaryotes = 28S rRNA
    *Environment where no protein, only rRNA
    *Early forms of life associated with rRNA catalysed reactions
44
Q

What is the role of eEF2?

A

eukaryotic Elongation Factor 2 = G-protein
- Involved in elongation of translation
- Catalyses translocation of the ribosome along mRNA
- Ensures proper alignement of codon and anti-codon on ribosomal sites
- Bound to E site → faciliates movement of tRNA from P to E site
When eEF2-GTP hydrolysed → translocation tightly regulated

45
Q

Why is it important that hydrolysis of eEF2-GTP be tightly regulated?

A

If ribosome moves by more than one codon on mRNA, can cause mutation in amino acid sequence, also dictates the rate of translation

46
Q

When/How is tRNA pushed out of the E site?

A

EF1-alpha release + GTP hydrolysis of the next amino acids coming into the A site → ribosomal conformational change → forces tRNA out of the ribosome
*At this moment tRNA is not linked to its amino acids anymore
tRNA gets recycle
*Growing polypeptide chain is always covalently linked to one of the tRNAs in P or A site

47
Q

How does translation termination occur?

A
  • Involves termination factors that recognise the stop codon (No tRNA has an anti-codon loop that matched the stop codon)
    1. eRF1 associated with GTP-eRF3
    2. Stop codon recognized by eRF1 (structurally mimic aminoacyl-tRNAs)
    3. GTP-eRF3 hydrolyzed → promotes hydrolysis of ester bond between last tRNA in P site and its amino acid → freeing of the polypeptide chain → post-termination complex
    4. Translocation of last tRNA to E site → exit
    5. Post-termination complex actively dissociated → large and small subunit ready to initiate another cycle
48
Q

What is included in the post-termination complex?

A
  • Polypeptide chain has left the Ribosome
  • last tRNA still interacting with P site
  • E site empty, ready to receive last tRNA
  • A site = mRNA stop codon interacting woth eRF1 (associated with eRF3-GDP) NOT eRF3-GTP, already hydrolysed bc polypeptide chain has been freed
49
Q

after the post-termination complex, what state does ribosome take?

A

Large and Small subunit dissociate
- Small subunit: reassociates with initiation factors eIF3 and eIF1 on E site and eIF1A on A site

50
Q

What is a polysome?

A

Complex formed by multiple ribosomes translating 1 single mRNA molecule → rapid and efficient synthesis of proteins

51
Q

What can centrifugation tell use about polysomes?
What can we see with sedimentation?

A

Can be used to quantify translational activity/efficiency of different mRNAs

When sedimentation:
At top → Bottom:
Initiation factors → 40S (small ribosomal subunit) → 60S (large ribosomal subunit) → 80S (fully associated ribosome) → Polysomes from less translated to more translated
(more translated at the bottom bc more ribosomes on the RNA so heavier)

52
Q

Which region of the Cargo protein that has to be imported to the nucleoplasmic side binds to importin?

A

The NLS (Nuclear Localizing Signal)

53
Q

Which RNA pol is responsible for synthesis of snRNA? tRNA? of 5S rRNA?

A

BIOL 200 (11 decks)

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