Translation

Question 1

Stop Codons

Answer 1

UGA, UAA, UAG

Question 2

First Proofreading Step of Translation

Answer 2

Happens within the tRNA, there is editing and synthesis site. If wrong AA is incorporated into synthesis site, AA is incorporated into editing site

Question 3

Formation of Pre-Initiation Complex

Step 1: ____ puts Met at the beginning of the protein, binds to ____, which binds to _____. This all binds to ____ subunit.

Cap of mRNA is bound to _____, which binds _____, which has interactions with _____, circularizing mRNA, making it a good substrate for other initiation factors to bind to 5’ end of mRNA.

Answer 3

Step 1: itRNA puts Met at the beginning of the protein, binds to eIF2, which binds to GTP. This all binds to small subunit.

Cap of mRNA is bound to eIF4E, which binds eIF4G, which has interactions with poly-a BPs, circularizing mRNA, making it a good substrate for other initiation factors to bind to 5’ end of mRNA.

Question 4

EIF2

Answer 4

Binds to GTP and itRNA
Complex binds to small ribosomal subunit in pre-initiation complex
Delivers initiator tRNA
-bac counterpart is IF2

Question 5

EIF4E

Answer 5

Binds cap of mRNA, which binds EIF4G

-no bacterial counterpart

Question 6

EIF4G

Answer 6

Binds EIF4E and poly-A BPs, circularizing mRNA, making it a good substrate for other initiation factors to bind to 5’ end of mRNA
-no bacterial counterpart

Question 7

What happens when small ribosomal subunit scans for AUG codon?

Answer 7

ATP is expended to allow for helicase activity

Question 8

What happens when 60S large subunit joins?

Answer 8

EIF2 and other initiation factors dissociate

Hydrolysis of GTP, causing EIF2 release

Question 9

Step 1 of Elongation

Delivery of aa-tRNA to _____ site and ____ site release
-____ is bound to GTP, and _____, which can basepair to mRNA via anticodon loop

Answer 9

Delivery of aa-tRNA to A side and E site release

-EEF1A is bound to GTP, and aminoacyl transferase, which can basepair to mRNA via anticodon loop

Question 10

Step 2 of Elongation

GTP Hydrolysis and ____ release, which ensures fidelity, that the right tRNA is there, ribosome proofreads in A site, where codon/anticodon pairs are checked by ribosome conformation

Answer 10

GTP Hydrolysis and eEF1A release, which ensures fidelity, that the right tRNA is there, ribosome proofreads in A site, where codon/anticodon pairs are checked by ribosome conformation

Question 11

Step 3 of Elongation

_____ promotes sliding of large subunit to next codon, with GTP attached
Catalyzes ______

Answer 11

EEF2 promotes sliding of large subunit to next codon, with GTP attached
Catalyzes TRANSLOCATION

Question 12

Step 4 of elongation

GTP hydrolysis and ____ release, marking completion of cycle

Answer 12

GTP hydrolysis and eEF2 release, marking completion of cycle

Question 13

EEF1A

Answer 13

Binds all canonical tRNAs except for iTRNA and selenocysteine tRNA
G protein
-bacterial counterpart is EFTu

Question 14

EEF2

Answer 14

G protein that is required for ribosome translocation

-EFG is bacterial counterpart

Question 15

Termination

Answer 15

ERF1 looks like a tRNA, binds A site, promotes release of completed polypeptide, ribosomal subunits dissociate
-NO TERMINATOR tRNA

Question 16

Differences between bacterial and eukaryotic translation

Answer 16

• Transcription and translation are coupled in bac because there is no nuclear membrane
• mRNAs are polycistronic in bac
• No 5’ cap is necessary in bac
• In bac, shine-dalgarno sequence is upstream of start codon, which directly bps to ribosomal RNA
• Bac ribosomes are smaller and divergent from mammalian ribosomes to allow selective inhibition by antibiotics
• Ribosome inhibitors typically do have residual toxicity due to theit effects on mitochondrial ribosomes

Question 17

Apo B editing

Answer 17

Intestinal (B-48) is shorter, C gets deaminated to U (enzymatically), creating premature stop codon

Question 18

miRNA

Answer 18

Inhibit translation via usually binding to 3’ UTR
RISC complex is thought to physically impede translation initiation
-many variations of bping bw miRNA and mRNA make target identification difficult

Question 19

IRE and IRP

Answer 19

• in low [Fe], IRP binds IRE and prevents formation of initiation complex, halting translation of ferritin (Fe storage protein)
• in high [Fe], IRP cannot bind IRE because IRP is bound to Fe, so Fe changes the protein’s conformation

Question 20

Phosphorylation of eIF2 Leads To….

Answer 20

Translation inhibition in response to

1. Low [AA] due to poor nutrition
2. Cellular stress
3. Immune response
4. Unfolded proteins

Question 21

eIF2 recycling requires…

Answer 21

eIF2B GEFs because affinity for GDP is much higher for than that for GTP

Question 22

The Different Nutritional States and Regulation of eIF2

Normal conditions: GDP is exchanged for GTP via ______, causing active eIF2

Poor nutrition conditions: _____ phosphorylates eIF2, which sequesters all of eIF2B as an inactive complex, which _______.

Answer 22

Normal conditions: GDP is exchanged for GTP via eIF2b, causing active eIF2

Poor nutrition conditions: protein kinase phosphorylates eIF2, which sequesters all of eIF2B as an inactive complex, which slows protein synthesis dramatically

Question 23

How Hypoxia Regulates Translation

Answer 23

mTOR signaling pathway represses translation in response to hypoxia by regulating funciton of eIF4E.

mTOR pathway is like a cellular rheostat, it up-regulates translation during growth and down-regulates during stress

mTOR is constitutively active

Normoxia: induces mTOR signaling
-mTOR is a kinase, it phosphorylates 4EBP, which keeps 4EBP from interfering with translation

Hypoxia: mTOR does not phosphorylate 4EBP, so it binds 4E, precluding translation

Question 24

Selenocysteine Incorporation and it’s importance

Answer 24

Requires tRNA, SBP2, eEFSec, UGA codon, SECIS element in 3’UTR

• selenocysteine is encoded by UGA stop codon that has been re-coded to allow Sec-tRNA binding
• SBP2 protein is required for recoding process
• Some rare forms of hypothyroidism are caused by SBP2 mutations in humans because reduced Sec incorporation leads to reduced production of deiodinases

Question 25

Ribosomal Frameshifting

Answer 25

A single mRNA can encode more than one protein product if the ribosomes are signaled to frameshift

• Frameshift signals include RNA pseudoknot and RNA sequence called ‘slippery site’
• Efficiency of frameshifting determines the amount of each protein made

Question 26

Poliovirus IRE

• Poliovirus shuts down host translation by expressing _____ that cleaves ______
• Poliovirus ____ binds to cleaved version of _____ and recruits the _______ directly, without the need for a cap.
• This system permits efficient viral mRNA translation at the expense of host mRNA translation

Answer 26

• Poliovirus shuts down host translation by expressing PROTEASE that cleaves eIF4G
• Poliovirus IRES binds to cleaved version of eIF4G and recruits the PRE-INITIATION COMPLEX directly, without the need for a cap.
• This system permits efficient viral mRNA translation at the expense of host mRNA translation

Question 27

Diptheria Toxin

Answer 27

• B subunit of toxin binds receptor on cell membrane, causing endocytosis
• The dimer dissociates
• A subunit goes into cytosol, catalyzes transfer of ADPR from NAD+ to EF-2, causing it to halt translation
• modifies elongation factor responsible for translocation

Question 28

Aminoacyl Synthetases

Require: ____
Recognize: _____ and _________
Has ____ site and _____ site

Answer 28

Aminoacyl Synthetases

Require: ATP
Recognize: AA and ANTICODON OF tRNA
Has SYNTHESIS site and EDITING site