A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.

The formation of a stable 43 S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3, and the eIF2-GTP-Met-tRNAi ternary complex (TC) in stabilizing the 43 S PIC are poorly defined. We have reconstituted the human 43 S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A, and eIF3j in the presence of different combinations of 43 S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43 S PIC components with the 40 S subunit. Human eIF1 and eIF1A bind cooperatively to the 40 S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A, and TC with the 40 S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40 S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40 S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40 S subunit. Together, our data provide a more complete thermodynamic framework of the human 43 S PIC and reveal important interactions between its components to maintain its stability.

In the scanning model of translation initiation, the decoding site and latch of the 40S subunit must open to allow the recruitment and migration of messenger RNA (mRNA); however, the precise molecular details for how initiation factors regulate mRNA accommodation into the decoding site have not yet been elucidated. Eukaryotic initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA entry channel and A-site of the 40S subunit. Previous studies have shown that a reduced affinity of eIF3j for the 43S preinitiation complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j affinity likely reflects a state of full accommodation of mRNA into the decoding site. Here, we have used a fluorescence-based anisotropy assay to quantitatively determine how initiation components coordinate their activities to reduce the affinity of eIF3j during the recruitment of mRNA to the 43S PIC. Unexpectedly, we show that a full reduction in eIF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of eIF4A. This result suggests that in addition to its helicase activity, eIF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during mRNA recruitment. Therefore, our results define eIF4A as a universal initiation factor in cap-dependent translation initiation that functions beyond its role in RNA unwinding.

The eukaryotic translation pathway has been studied for more than four decades, but the molecular mechanisms that regulate each stage of the pathway are not completely defined. This is in part because we have very little understanding of the kinetic framework for the assembly and disassembly of pathway intermediates. Steps of the pathway are thought to occur in the subsecond to second time frame, but most assays to monitor these events require minutes to hours to complete. Understanding translational control in sufficient detail will therefore require the development of assays that can precisely monitor the kinetics of the translation pathway in real time. Here, we describe the translation pathway from the perspective of its kinetic parameters, discuss advances that are helping us move toward the goal of a rigorous kinetic understanding, and highlight some of the challenges that remain.

The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to occur by way of a ternary complex (TC) comprising eIF2, GTP and Met-tRNA(i). We have generated from purified human proteins a stable multifactor complex (MFC) comprising eIF1, eIF2, eIF3 and eIF5, similar to the MFC reported in yeast and plants. A human MFC free of the ribosome also is detected in HeLa cells and rabbit reticulocytes, indicating that it exists in vivo. In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the same rate as the TC. However, MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting that MFC components may play a role in the release of eIF2-GDP from the ribosome following AUG recognition. Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly together with the TC. However, the MFC protein components also bind individually to 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor complexes. Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are identified, but which one predominates in vivo remains to be elucidated.

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.

A key step in translational initiation is the recruitment of the 43S preinitiation complex by the cap-binding complex [eukaryotic initiation factor 4F (eIF4F)] at the 5' end of messenger RNA (mRNA) to form the 48S initiation complex (i.e., the 48S). The 48S then scans along the mRNA to locate a start codon. To understand the mechanisms involved, we used cryo-electron microscopy to determine the structure of a reconstituted human 48S The structure reveals insights into early events of translation initiation complex assembly, as well as how eIF4F interacts with subunits of eIF3 near the mRNA exit channel in the 43S The location of eIF4F is consistent with a slotting model of mRNA recruitment and suggests that downstream mRNA is unwound at least in part by being "pulled" through the 40S subunit during scanning.

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases^1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.