8_16_16 Translation

Question 1

Basics of protein synthesis

Answer 1

last opportunity for regulation

rapid response to stimuli as compared to transcription

many pharmaceuticals affect translation

Question 2

mRNA

Answer 2

degenerate nucleotide codons code for 20 amino acids

typical mammalian mRNA

5’ cap made during elongation, regulatory 5’ untranslated region, start codon (AUG), coding region, stop codon (UAA, UGA, UAG), long 3’ UTR (primary site of regulation and conformation info), poly A tail

insertions and deletions typically introduce premature stop codons

Question 3

tRNA

Answer 3

affect the translation reaction (aminoacylation)

amino acid attached to post transcriptionally added CCA sequence at 3’ end also contains three loops (D, anticodon, T)

process: ATP dependent attachment if aa to tRNA via Aminoacyl-tRNA sythetase, heavily regulated with one variety of tRNA for each a.a. tRNA checked by modulating from a editing and synthesis site on the above enzyme

Question 4

Ribosome

Answer 4

made of a combo of proteins and rRNAs, two major subunits (60S and 40S), activity is done entirely by the rRNA, the tRNA wedges between subunites and progresses through three sites APE (aminoacyl, peptidyl and exit)

Question 5

Initiation

Answer 5

Step 1: formation of the pre-initiation complex
eIF2-GTP delivers Met-tRNA to the small ribosomal subunit …mRNA with the 5’ end in eIF4E and the poly-A chain bound to eIF4G loop increasing affinity comes in with additional initiation factors… the 5’ mRNA-eIF4E/G complex is recognized and binds to the small ribosomal subunit to form the pre-initiation complex

Step 2: eIF4EandG stay at 5’ end of mRNA and the rest scans for AUG start codon in ATP dependent RNA helicase activity

Step 3: At start codon eIF2 facilitates GTP hydrolysis and releases from the pre-initiation complex allowing large ribosomal subunit to hop on

Question 6

Initiation specifics to know

Answer 6

eIF4E binds 5’ cap

eIF2 binds and delivers Met-tRNA (needs GTP)

eIF4G- scaffolds eIF4E and mRNA in conformation to bind to pre-initiator complex

initiator tRNA is unique not used for translated methionine

GTP hydrolysis releases eIF2

GTP hydrolysis is common to signal next steps

Question 7

Elongation

Answer 7

Step 1: eEF1A-GTP comes in with a tRNA-AA and deposits it in the A site of the ribosome as old tRNA from E site leaves

Step 2: eEF1A release mediated by GTP hydrolysis, self reg. of mistakes by strength of codon-anticodon interaction

Step 3: eEF2-GTP binds to A-site, GTP hydrolysis ratchets subunits with release of eEF2

Question 8

Elongation need to know

Answer 8

eEF1A binds all canonical tRNAs

GTP hydrolysis required for release and ratcheting

eEF2 is another G protein needed for translocation

proofreading in the A site by ribosome conformation

Question 9

Termination

Answer 9

eERF1 (tRNA analog) binds in A site, regulated by G-protein eRF3, catalyzes peptide release

Question 10

post-transcriptional regulation of translation

Answer 10

C to U deamination introduces early stop codon to make different enzyme lengths for different systems

miRNA bind nRNAs and inhibit translation (lots in development) through formation of RIS-Complexes

Iron homeostasis mediated by Iron Sensing Protein (ISP) for ferritin translation (atypically, this is on 5’ UTR)

Phosphorylation of eIF2 due to malnutrition, cell distress, an immune response or ER issues can limit translation, this protein normally Guanidine Exchange Factor for eIF2, when phosphorlylated, the initiation complex can’t form

hypoxia too (mTOR pathway regs eIF4E, huge pathway that both promotes and represses)
Under normoxia active mTOR phosphorylates 4EBP-1 allowing eIF4E to do it's pre-initiator complex thing
hypoxia: it binds stopping eIF4E from binding the eIF4G

Question 11

Diseases of misregulation of translation

Answer 11

hypothyroidism from selenocysteine deficiency: serine enzymatically converted to SC while in complex with tRNA and coded for by the stop codon UGA, often incorporates into redox regulatory proteins especially for TH synthesis - has specially elongation factor, tRNA, element in 3’UTR, regulator binding protein for binding ternary complex

Ribosomal frameshifting
polyX slippery sites causes reading frame shifts to make more than one protein from one mRNA in pseudoknots
AIDS

polio targets eIF4G stopping some host initiation

diptheria stops elongation by messing with eEF2