Michael G. Acker

Development and characterization of a reconstituted yeast translation initiation system.

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

RECONSTITUTION OF YEAST TRANSLATION INITIATION

To facilitate the mechanistic dissection of eukaryotic translation initiation we have reconstituted the steps of this process using purified Saccharomyces cerevisiae components. This system provides a bridge between biochemical studies in vitro and powerful yeast genetic techniques, and complements existing reconstituted mammalian translation systems (Benne and Hershey, 1978; Pestova and Hellen, 2000; Pestova et al., 1998; Trachsel et al., 1977). The following describes methods for synthesizing and purifying the components of the yeast initiation system and assays useful for its characterization.

Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining.

Eukaryotic initiation factor 5B (eIF5B) is a GTPase that facilitates joining of the 60 S ribosomal subunit to the 40 S ribosomal subunit during translation initiation. Formation of the resulting 80 S initiation complex triggers eIF5B to hydrolyze its bound GTP, reducing the affinity of the factor for the complex and allowing it to dissociate. Here we present a kinetic analysis of GTP hydrolysis by eIF5B in the context of the translation initiation pathway. Our data indicate that stimulation of GTP hydrolysis by eIF5B requires the completion of early steps in translation initiation, including the eIF1- and eIF1A-dependent delivery of initiator methionyl-tRNA to the 40 S ribosomal subunit and subsequent GTP hydrolysis by eIF2. Full activation of GTP hydrolysis by eIF5B requires the extreme C terminus of eIF1A, which has previously been shown to interact with the C terminus of eIF5B. Disruption of either isoleucine residue in the eIF1A C-terminal sequence DIDDI reduces the rate constant for GTP hydrolysis by ∼20-fold, whereas changing the aspartic acid residues has no effect. Changing the isoleucines in the C terminus of eIF1A also disrupts the ability of eIF5B to facilitate subunit joining. These data indicate that the interaction of the C terminus of eIF1A with eIF5B promotes ribosomal subunit joining and possibly provides a checkpoint for correct complex formation, allowing full activation of GTP hydrolysis only upon formation of a properly organized 80 S initiation complex.

Kinetic analysis of late steps of eukaryotic translation initiation.

Little is known about the molecular mechanics of the late events of translation initiation in eukaryotes. We present a kinetic dissection of the transition from a preinitiation complex after start codon recognition to the final 80S initiation complex. The resulting framework reveals that eukaryotic initiation factor (eIF)5B actually accelerates the rate of ribosomal subunit joining, and this acceleration is influenced by the conformation of the GTPase active site of the factor mediated by the bound nucleotide. eIF1A accelerates joining through its C-terminal interaction with eIF5B, and eIF1A release from the initiating ribosome, which occurs only after subunit joining, is accelerated by GTP hydrolysis by eIF5B. Following subunit joining, GTP hydrolysis by eIF5B alters the conformation of the final initiation complex and clears a path to promote rapid release of eIF1A. Our data, coupled with previous work, indicate that eIF1A is present on the ribosome throughout the entire initiation process and plays key roles at every stage.

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

Mechanism of ribosomal subunit joining during eukaryotic translation initiation

Decades of research have yielded significant insight into the mechanism by which a cell translates an mRNA into the encoded protein. However many of the molecular details of the process remain a mystery. Translation initiation is an important control point in gene expression, and misregulation can lead to diseases such as cancer. A better understanding of the mechanism of translation initiation is imperative for the development of novel therapeutic agents. Recently, a combination of genetic, biochemical and biophysical studies has begun to shed light on how, at a molecular level, the translational machinery initiates protein synthesis. In the present review, we briefly compare and contrast the initiation pathways utilized by bacteria, archaea and eukaryotes, and then focus on translation initiation in eukaryotes and recent advances in our understanding of the subunit joining step of the process.

rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

ABSTRACT The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

Structural integrity of α-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability

Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factors binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNA(i)(Met) bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding.

Intragenic Suppressor Mutations Restore GTPase and Translation Functions of a Eukaryotic Initiation Factor 5B Switch II Mutant

ABSTRACT Structural studies of GTP-binding proteins identified the Switch I and Switch II elements as contacting the γ-phosphate of GTP and undergoing marked conformational changes upon GTP versus GDP binding. Movement of a universally conserved Gly at the N terminus of Switch II is thought to trigger the structural rearrangement of this element. Consistently, we found that mutation of this Gly in the Switch II element of the eukaryotic translation initiation factor 5B (eIF5B) from Saccharomyces cerevisiae impaired cell growth and the guanine nucleotide-binding, GTPase, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement for this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element and at a residue in domain II of eIF5B that interacts with Switch II. The intragenic suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain II suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis and thereby reestablish coupling between GTP binding and eIF5B domain movements.

New techniques for the study of cardiomyoplasty

Despite a paucity of objective hemodynamic evidence, cardiomyoplasty has been shown to subjectively improve functional status and symptoms. In addition, the factors associated with successful cardiomyoplasty have not been defined. We have been using magnetic resonance techniques to quantify stroke volume and the three-dimensional motion of the heart during cardiomyoplasty. We have also been studying the regional mechanics and blood flow of partially isolated latissimus dorsi to determine whether the force generation and shortening of the proximal portion of the latissimus dorsi (that have typically been measured) are representative of the distal portion that is in contact with the heart.