Term
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Definition
Proteins that help in import ("importins") and export ("exportins") through the nuclear pore. They recognize nuclear export signals (exportins) and nuclear localization signals (importins). These signals can be peptides or RNA
A common feature of these proteins is that they interact with Ran protein (a GTPase, whose GTP/GDP status may direct it's role as importer/exporter)
NOTE: remember Karyo- as the root for nucleus, coming from the greek karyon meaning "NUT" |
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Term
| Which exportin does tRNA bind after it's processed? |
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Definition
| exportin-t. This binding is dependent on proper tRNA structure. |
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Term
Which form of Ran gets exported from the nucleus? Imported?
Can you name some proteins involved in these steps? |
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Definition
Ran is the protein that works with Karyopherins to export/import shit.
the GTP-bound form of Ran is exported; the GDP bound form is imported
RanGAP and RanBP1/2 are cytoplasmic & induce GTPase activity
RanGEF and RCC1 are nuclear and induce GTP binding |
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Term
Briefly describe methods of nuclear export for:
-tRNA
-miRNA
-snRNA
- mRNA |
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Definition
- tRNA is bound by exportin-t when it has folded correctly. Exportin-t binds Ran-GTP --> exported
- miRNA is bound by exportin-5 --> exportin-5 binds Ran-GTP --> export
- CBC ("cap-binding complex) binds snRNA and PHAX (a NES-containing protein) --> PHAX is bound by exportin-1 (aka CRM1) --> Ran-GTP binds CRM1 --> export
- recognized by TAP/MEX67 & uses either ALY/REF or a signal-recognition (SR) protein; (NOTE: ALY/REF binds a helicase that is part of the splicing machinery to localize the complex to the mRNA) |
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Term
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Definition
| localized to cytoplasmic face of nuclear pore (cytoplasmic fibers) and is thought to be involved in the removal of proteins from RNPs as they exit the nuclear pore |
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Term
| name 3 advantages of RNA localization |
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Definition
1) spacially-restricted gene expression
2) local stimuli can regulate "on sites" without having to communicate with the nucleus
3) transporting 1 mRNA to make many proteins is more efficient than transporting all the proteins individually |
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Term
MOA and stimulator/inhibitor of:
1) Ccr4p
2) PAN2/PAN3
3) PARN trimer |
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Definition
All 3 are responsible for degrading mRNA
1) Ccr4p has deadenylating properties (complexed with Pop2). Part of the machinery that degrades mRNA. Inhibited by PAB protein
2) important in Ccr4 mutants. Enhanced by PAB protein. Important for initial poly-A truncation to 55-75 nt
3) Inhibited by PAB, stimulated by 5' cap ; principal deadenylase in mammals (absent in yeast and flies) |
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Term
| which end of the mRNA does the exosome degrade from? What is the RNase? |
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Definition
| 3'-->5'; RNase activity contained within Rrp44; Thought to function by mRNA being fed through ring structure to Rrp44, which chops it up |
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Term
how does PABP inhibit mRNA decapping?
What degrades mRNA after decapping has occured? |
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Definition
PABP binds m7G cap and prevents DCP1/DCP2 from binding and degrading the mRNA cap.
XRN1 |
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Term
For bacterial ribosomes, what name subunits and their constituents
Same for eukaryotes |
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Definition
50S: 5S & 23S rRNAs + 33 proteins
30S: 16S rRNA + 21 proteins
These make up the 70S ribosome
60S: 5S, 5.8S, 25S rRNA + 45 proteins
40S: 18S rRNA + 32 proteins
make up 80S ribosome |
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Term
| When does ribosomal assembly occur? (i.e. during or after transcription) |
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Definition
| during transcirption (proceeds in 5'->3' direction, probably to avoid kinetic traps); however, people can assemble ribosomes in vitro by manipulating temperature and time. |
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Term
| Describe the genomic organization of ribosomes and their corresponding mRNA and processing |
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Definition
They are transcribed as a single RNA with spacer sequences between rRNA segments. After transcription, many rounds of cleavage by different RNases eventually form the different rRNA subunits (e.g. RNase P,E,H,III)
The final cleavage of 18S occurs in the cytoplasm by Nob1, however Nob1 was loaded in nucleus |
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Term
| What is U3 snRNA used for? |
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Definition
| U3 is used to help 18S form its pseudoknot (NOTE, this is the only "U_" RNA to not be part of the splicing machinery) |
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Term
| snoRNAs play what part in ribosome assembly/ |
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Definition
They modify ribosomes (e.g. rotate nucleotides); many of these nucleotide modification are located near the active site of the ribosome. 30S can be active w/out modificiation, however the activity is reduced
stand for "small nucleolar RNAs" |
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Term
| what does Efl1 do during ribosome biogenesis? |
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Definition
Efl1 is a paralog of eEF2 (the translocase), it "test-drives" the ribosome
defects in the "test drive" (e.g. Rpl10 & Sdo1) are responsible for hereditary syndromes (T-cell ALL, Swachman-diamond syndrome) |
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Term
| what are the sites in the ribosome and what doe each of them hold? |
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Definition
| A-P-E sites. A site holds incoming tRNA (peptide chain transferred here transiently); P site holds growing peptide chain; E site ejects empty tRNA from P site after it transfers chain to A site |
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Term
| how is the amino acid transfered to tRNA |
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Definition
| first, it is connected to ATP + AA -> AMP-AA (5' linkage) + PPi; then, a terminal A on tRNA nucleophilically attacks with 2' hydroxyl to transfer the AA to it (2' linkage) releasing AMP |
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Term
| where does Aminoacyl tRNA synthase contact the tRNA |
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Definition
it contacts it widely (must be sure it's putting the correct AA on the correct tRNA), importantly it contacts it in the acceptor stem, D-stem, and anticodon loop (first and last are intuitive, but D-stem is also specific)
it must also recognize the |
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Term
| how many proteins can 1 mRNA code for (species specific or no?) |
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Definition
| in eukaryotes, 1mRNA = 1protein; in prokaryotes, 1mRNA = many proteins (many ORFs) |
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Term
| what is the Shine-Dalgarno sequence and what binds to it? |
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Definition
| Translation starts just after it. The 16S rRNA binds to it |
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Term
| What is the cap-binding complex that stimulates translation of capped mRNAs? |
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Definition
eIF4F, made up of 3 proteins:
eIF4E: binds cap directly
eIF4G: large (220kDa) protein that binds many other proteins
eIF4A: a DEAD-box helicase that is stimulated by eIF4B |
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Term
| Give 3 reasons why it's probably beneficial that PABP binds eIF4G |
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Definition
1) Selects for full-lenght mRNAs
2) prevents degradation
3) Ribosomes are close to initiation site after they finish translation
(possible 4th: regulator proteins bind in 3' UTR, thus they are put into proximity of the start codon) |
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Term
| describe how reticulocytes regulate gene expression in Fe depleted environment? |
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Definition
| low [Fe] -> regulator protein phosphorylates eIF2alpha -> can't release eIF2B -> can't initiate translation |
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Term
What delivers the aa-tRNA to the ribosome?
What is a ternary complex wrt a ribosome?
What is the MOA of streptomycin? |
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Definition
EF-tu, a GTPase
ternary complex is EF-Tu + aa-tRNA + GTP; The ternary complex delivers aa-tRNA to the ribosome, then the ribosome-dependent GTPase activity of EF-Tu hydrolyzes GTP and the EF-Tu + GDP complex dissociates to participate in another round.
Streptomycin interferes with the proofreading ability of the ribosome; It locks it in the "ram" state (note: there are 2 states, "ram" and "restrictive", the ram is fast but inaccurate) |
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Term
what is the function of EF-G?
EF-P? |
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Definition
EF-G has an analogous role as EF-Tu, but instead of delivering a tRNA, it causes the A-site tRNA to move to the P site
EF-P nudges the Met-tRNA from the E-site to the P-site during initiation |
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Term
| how were releasing factors discovered (i.e. what was the experiment?) wrt ribosomes? |
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Definition
| f-MET was bound to AUG followed by a stop codon (UAA, UAG, UGA) -> different ribosomal fractions were added and fMET release was measured |
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Term
| how do eukaryotic cells determine if htere is a premature stop codon (i.e. mechanism of nonsense mediated decay) |
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Definition
splicing leaves exon junction complexes (EJCs) -> if a stop codon is detected upstream of these, the mRNA is targeted for degradation (via decapping & 5'->3' degradation, which takes place in the presence of a poly-A tail!))
UPF1/UPF2 are part of the EJC, they detect a stop codon upstream of the EJC and activate NMD. UPF1/2 become phosphorylated and bind to eIF3 to prevent conversion of 48S initiation complex to the 80S complex. Since the ribosome cannot translate, the mRNA is degraded (probably using P-bodies)
NOTE: cricket paralysis virus, which has an IRES that is eIF3-independent is not subject to nonsense-mediated decay |
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Term
| describe how reticulocytes regulate gene expression in Fe depleted environment? |
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Definition
| low [Fe] -> regulator protein phosphorylates eIF2alpha -> can't release eIF2B -> can't initiate translation |
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Term
| name the 3 common elements between siRNA and miRNA |
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Definition
1) dsRNA trigger
2) Dicer processing
3) Argonaute (Ago) containing complex
NOTE: non-optimal bp between RNAi and target mRNA will use miRNA (i.e. non-degradative) pathway more readily than siRNA (i.e. degradative) pathway |
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Term
| name components of argonaute. How does siRNA get delivered to it? |
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Definition
Argonaute has 4 domains, but 2 are important:
PAZ domain binds ss siRNA
PIWI domain cleaves dsRNA (slicer activity)
RISC Loading Complex (RLC) must deliver the siRNA to Ago2. RLC includes dicer & R2D2. RLC determines which strand will be the guide strand by Dicer binding the less stable end of the dsRNA. The 5' strand at this end will become the guide strand. After Dicer has cut, Ago2 cuts the non-guide strand and it dissociates |
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Term
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Definition
| short hairpin RNA. They are engineered siRNA to have hairpins. Transformed cells expressing shRNAs provide a constant supply of dsRNA for siRNA knockdown, as opposed to transforming dsRNA (which is very short lived in mammalian cells, and therefore not too useful in experiments) |
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Term
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Definition
| Argonaut can function in the siRNA pathway to silence genes at the DNA level: guide strand binds nacent mRNA -> Ago stimulates histone methyltransferase or DNA methylation (i.e. formation of heterochromatin leading to gene silencing) |
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Term
| when does miRNA cleave? block translation? |
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Definition
cleaves when miRNA has perfect/near-perfect basepairing to target mRNA (acts like siRNA)
Blocks translation when it has imperfect base pairing |
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Term
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Definition
protein involved in miRNA silencing via mRNA-mediated degradation. It interacts with the Ago complex (bound near 3' end), however, it can be Ago-independent if it is tethered to the mRNA
GW182 induces loss of poly-A tail, which releases the looped mRNA and activates the decapping machinery |
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Term
where does SRP hang out? (and what is it?)
What are the other proteins that function (independently of SRP) in the same pathway? |
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Definition
SRP ("signal recognition particle") is an RNP that recognizes export signals. it sits on the ribosome and stops translation when an SRP comes its way (in eukaryotes, in prokaryotes, it doesn't stop translation)
SRP delivers the ribosome to the SRP receptor, which in turn connects to SecY (The translocation channel)
SecB (chaperone) and SecA . They use same pore as SRP, thought (SecYEG) |
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Term
| What organisms have a Shine Dalgarno sequence (& what is it?) |
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Definition
| prokaryotes have a shine-dalgarno sequence; it initiates translation |
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Term
| what protein "test-drives" translocation in eukaryotic ribosomes? What is its paralog in normal translation? What is its energy source? |
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Definition
| Efl1; paralog of eEF2 (aka EF-G); uses GTP, just like EF-G |
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