Regulation of Protein Translation By Emmanuel Landau

Why Regulate Translation? Produces proteins rapidly Produces proteins locally Produces single proteins or classes of proteins BUT It is very costly in energy.

There are two overall mechanisms of translation control. Regulation by modifying proteins Regulation using micro RNAs (miRNA’s)

Mechanism of Translation

It is organized in two subunits: Translation generates proteins according to the instructions read from messenger RNA (mRNA). mRNA is translated by moving through a groove in the ribosome. The ribosome is a multicomponent entity, composed of ribosomal RNA’s and 78 different proteins. It is organized in two subunits: A 40S and a 60S subunit.

Phases of Translation Translation is divided into two stages: initiation and elongation. Initiation brings the mRNA to the ribosome and uses a large number of initiation factors to assemble the ribosome and begin translation. Elongation then continues to assemble amino acids to form the protein.

Sequence of events leading to translation initiation Sonenberg et al., eds., Translational Control of Gene Expression (2000), p. 46.

Initiation Step 1. Capture of mRNA The 5’ cap (m7GpppX) of the mRNA is captured by binding to a complex of eukaryotic Initiation Factors of the eIF4 family. These are eIF4G, a scaffold protein; eIF4E, which is bound to eIF4G and holds the mRNA; and eIF4A and eIF4B, which unravel kinks in the mRNA. The four eIF4’s (G,A,B,E) are collectively known as eIF4F.

The Binding of eIF4F to the 5’ Cap of mRNA Modified from Sonenberg et al., eds. Translational Control of Gene Expression (2000), p. 46.

Regulation of Step 1 1. Phosphorylation of the eIF4E binding proteins, the 4E-BPs. Why does this help? Because the 4E-BPs inhibit eIF4E’s function, and their phosphorylation liberates eIF4E from inhibition.

Phosphorylation of 4EBP Allows eIF4E Association with eIF4G Sonenberg et al. eds Translational Control of Gene Expression (2000) p. 247

Phosphorylation of 4EBP by mTOR and Upstream Pathway Sonenberg et al. eds Translational Control of Gene Expression (2000) p. 252

Regulation of Step 1 1. Phosphorylation of the eIF4E binding proteins, the 4E-BPs. 2. Binding of polyadenylate binding protein (PABP) to eIF4G. Why? Because this circularizes the polysome, and allows ribosomal subunits to start new ribosomes.

Lodish et al. Molecular Cell Biology Fig. 4-42 Polyadenylation and Circularization of mRNA Through Binding of PABP to eIF4G Lodish et al. Molecular Cell Biology Fig. 4-42

Circular mRNA Visualized by Atomic Force Microscopy eIF4E, eIF4G, and PABP are Present in the Light-Colored Regions Attached to each RNA Sonenberg et al. eds Translational Control of Gene Expression (2000) p. 454

In case of mRNAs with a CPE sequence in the 3’ end, the poly(A) tail also serves to disrupt the binding of maskin, a CPEB-binding protein to eIF4E. This makes eIF4E available to start building the cap- binding complex.

Modified from Groisman et al. Cell 109: 473 (2002) Polyadenylation Leads to the PABP-mediated Displacement of Maskin from eIF4E Modified from Groisman et al. Cell 109: 473 (2002)

Regulation of Step 1 1. Phosphorylation of the eIF4E binding proteins, the 4E-BPs. 2. Binding of polyadenylate binding protein (PABP) to eIF4G. 3. Phosphorylation of eIF4E allows it to detach from the cap and recycle.

MAPK-Dependent Phosphorylation of eIF4E Is Mediated by the eIF4G Associated Kinase Mnk Sonenberg et al. eds Translational Control of Gene Expression (2000) p. 270

Initiation Step 2: Assembly of the Preinitiation Complex Initiation factors 1, 1A, and 3 bind to the 40S ribosomal subunit. eIF2, activated by GTP and carrying Met-tRNA, joins the 40S complex. GDP-GTP exchange on eIF2 is enhanced by eIF2B, which acts as a GEF. eIF2 is inhibited by direct phosphorylation. mRNA-eIF4F now binds to 40S via eIF3.

Assembly of the Preinitiation Complex Modified from Sonenberg et al., eds. Translational Control of Gene Expression (2000), p. 46.

Initiation Step 3: mRNA Scanning, AUG Recognition and Ribosome Completion (40S+60S=80S) The preinitiation complex travels downstream along the 5’UTR of the mRNA until it arrives at the start codon (AUG) and recognizes it through interaction with the eIF2-GTP-tRNA complex. GTP is hydrolyzed by eIF5, the preinitiation complex unravels, the 60S subunit binds to the 40S subunit and translation begins.

Disassembly of the Preinitiation Complex Upon Recognition of the Start Codon (AUG) Modified from Sonenberg et al., eds. Translational Control of Gene Expression (2000), p. 46.

The Elongation Cycle (1) Translation starts with the AUG start codon positioned at the A site of the ribosome. As the ribosome continues to scan the mRNA, the AUG-tRNA-Met complex is translocated to the P site of the ribosome. A new AA-tRNA arrives at the vacated A site, courtesy of elongation factor 1A (eEF1A). eEF1A requires GTP for activation. Its GEF is eEF1B.

Elongation: Sequence of tRNA Displacements (A P E Sites) And Crystal Structures of the Ribosome Subunits Bacterial 30S and 50S Subunits of the Bacterial Ribosome are Shown, Complexed with EF-G (homologous to Eukaryotic 40S, 60S, and eEF2) Joseph, RNA 9:160 (2003).

Elongation Cycle (2a) The AUG-tRNA-met is now at the P site. The next available codon of the mRNA is now at the A site and the cognate aa-tRNA-eEF1A-GTP binds to it. The ribosome catalyzes a peptide bond between the methionine at the P site and the new amino acid. However, its tRNA is still at the A site.

Elongation: Successive AAs are Brought to the Vacant A-Site by Cognate tRNA Bound to eEF1A • GTP (E-Site not Shown; Nascent Protein at P-Site tRNA) Abbott and Proud, Trends Biochem.Sci. 29:25 (2004)

The Elongation Cycle (2b) 3. Elongation factor eEF2-GTP enters the ribosome, pushing the new tRNA into the P site, and the deacylated first tRNA into the exit (E) site. In the next cycle this tRNA will be ejected from the ribosome. 4. During this process GTP is hydrolyzed, and eEF2-GDP leaves the ribosome. 5. The cycle is repeated many times.

EF-G is Bacterial Homologue of eEF2 Binding of eEF2 • GTP to Ribosome Catalyzes tRNA Translocation from Small Subunit Binding Sites EF-G is Bacterial Homologue of eEF2 Joseph, RNA 9:160 (2003).

Translation can also be regulated by controlling the synthesis of translation factors. This process is mediated by the Ser/Thr kinase mTOR (mammalian target of rapamycin).

mTOR - p70-S6K Translation Pathway Proteins Involved in Translation NMDA-R PKC Protein Kinase A TRK-B MAP Kinase GPCRs mTOR - p70-S6K Translation Pathway 4E-BP Rapamycin Proteins Involved in Translation 5’ TOP mRNAs

Termination of Translation Releasing Factors (eRFs) are involved in termination. eRF1 structurally mimics tRNA that is bound to eEF1a • GTP. eRF1 fits into the ribosomal A-site, where it recognizes the stop codon. It then releases the completed polypeptide by catalyzing a nucleophilic attack on the ester bond between the peptide and the P-site tRNA. The catalytic activity of eRF1 is stimulated by the GTP-bound form of another relasing factor, eRF3.

Mimicry of tRNA by the Releasing Factor eRF1 Domain 2 on eRF1 Recognizes the Stop Codon Domain 3 Catalyzes the Hydrolysis of the Completed Peptide from the P-Site tRNA Song et al., Cell 100:311 (2000).

Conclusions Translation is regulated at all 3 phases: initiation, elongation, and termination. Initiation is the most highly regulated of these phases, involving a large number of initiation factors and accessory proteins. In addition to modification of translation factors either by phosphorylation or by GDP/GTP exchange, and the modification of mRNAs at their 3’ UTRs, protein synthesis can be controlled by the mTOR-mediated synthesis of additional translational machinery.