Molecular Biology

Chapter 17 Outline

Eukaryotic translation initiation:

∑ Translation starts at the first AUG that is in the context of a Kozak sequence; A Kozak sequence has the consensus CCRCCAUGG; Base alterations within the Kozak consensus reduce translation efficiency (F17.21); If an AUG in good Kozak context is upstream of the natural AUG, it will block translation (F17.22), but blocking can be eliminated by including a termination codon in frame with the first (blocking) AUG (F17.23); The second AUG is less efficiently used as the upstream open reading frame (ORF) gets longer; Weak RNA hairpins close to the cap can block translation, but weak hairpins further away from the cap have no effect on translation (F17.25); Strong RNA hairpins further downstream of the cap, but upstream from the AUG, block translation (F17.25); Some mRNAs (capped and uncapped) have internal ribosome entry sequences (IRESs) that enable translation of additional ORFs.

∑ Factors involved in translation initiation (F17.28).

a) eIF3 binds to the 40S ribosomal subunit and blocks premature binding of the 60S subunit.

b) eIF2 binds the initiating aminoacyl-tRNA to the 40S ribosomal subunit; Once the initiating aminoacyl-tRNA is bound, GTP is hydrolyzed to release eIF2; eIF2B is a guanine exchange factor that exchanges GTP for GDP on eIF2.

c) eIF4F is a cap binding protein that allows the 40S ribosome to bind mRNA; Cap binding is the rate limiting step in translation initiation; eIF4F is comprised of three major subunits, eIF4E, eIF4A and eIF4G (F17.31); RNA crosslinking studies showed that eIF4E binds to the 5π cap (F17.29); eIF4F stimulates translation (F17.30); eIF4A is an RNA helicase that unwinds RNA hairpins with the help of the ATP requiring factor eIF4B (F17.32); eIF4G is an adaptor protein that recruits 40S ribosomes to the mRNA and stimulate translation; eIF4G binds to eIF3, eIF4A and either eIF4E, PAB1p or an IRES recognition protein (F17.33).

d) eIF1/1A binds to 40S ribosome along with eIF3; eIF1/1A is necessary for the bound ribosome to scan to the Kozak AUG (F17.34); eIF1/1A stimulate formation of stable 48S complexes (where the ribosome has scanned to the AUG) by eliminating weak complexes at the point of binding the 43S complex to the mRNA (F17.36).

e) eIF5 promotes binding of the 60S ribosomal subunit to the 48S complex; ejects eIF2 and eIF3.

f) eIF6 binds to the 60S ribosomal subunit and dissociates the 80S ribosome.

Control of translation initiation in eukaryotes;

∑ Translation of hemoglobin is repressed when heme is in short supply; In the absence of heme, heme controlled repressor phosphorylates eIF2 so that it binds very tightly to eIF2B, thus blocking its ability to exchange GTP for other eIF2s (F17.38); Interferons also block translation of viral proteins by activating a kinase that phosphorylates eIF2.

∑ Phosphorylated eIF4E increases its cap binding affinity four fold; Insulin and other growth factors stimulate translation by removing a protein that blocks eIF4E (F17.39); The protein that blocks eIF4E is PHAS1 (rat) and 4E-BP1 (human); Insulin stimulates the release of 4E-BP1 (F17.41); Phosphorylation of PHAS1 causes its release from eIF4E (F17.43); mTOR1 is the kinase that phosphorylates PHAS1 (F17.39).

∑ When serum levels of iron are high, the translation of ferritin is stimulated; Ferritin contains an IRE in itπs 5πUTR; The IRE within the ferritin 5πUTR is responsible for iron dependent translation enhancement (F17.45); A repressor (IRE-binding protein) binds to the IRE and blocks translation until removed by binding to iron.