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Jon R Lorsch

Publications and source records attributed to Jon R Lorsch.

17 recordsLinked to original sources
PubMed2006

Intragenic suppressor mutations restore GTPase and translation functions of a eukaryotic initiation factor 5B switch II mutant.

Structural studies of GTP-binding proteins identified the Switch I and Switch II elements as contacting the gamma-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.

Amino Acid Substitution↗
PubMed2006

Where to begin? The mechanism of translation initiation codon selection in eukaryotes.

Selecting the codon at which to begin translation is a complicated event in an already complicated process. Many protein initiation factors (eIFs) have been implicated in start site selection, but the mechanistic details of their activities have remained obscure until recently. Biochemical and genetic studies of eIFs 1, 1A, 2 and 5 have suggested that the 43S pre-initiation complex exists in two conformations and that the changing interactions of the factors within the 43S pre-initiation complex in response to encountering an AUG codon regulates these conformations and, ultimately, the selection of the start codon.

Animals↗
PubMed2006

Initiation of protein synthesis by hepatitis C virus is refractory to reduced eIF2.GTP.Met-tRNA(i)(Met) ternary complex availability.

A cornerstone of the antiviral interferon response is phosphorylation of eukaryotic initiation factor (eIF)2alpha. This limits the availability of eIF2.GTP.Met-tRNA(i)(Met) ternary complexes, reduces formation of 43S preinitiation complexes, and blocks viral (and most cellular) mRNA translation. However, many viruses have developed counterstrategies that circumvent this cellular response. Herein, we characterize a novel class of translation initiation inhibitors that block ternary complex formation and prevent the assembly of 43S preinitiation complexes. We find that translation driven by the HCV IRES is refractory to inhibition by these compounds at concentrations that effectively block cap-dependent translation in vitro and in vivo. Analysis of initiation complexes formed on the HCV IRES in the presence of inhibitor indicates that eIF2alpha and Met-tRNA(i)(Met) are present, defining a tactic used by HCV to evade part of the antiviral interferon response.

Animals↗
PubMed2006

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation.

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.

Base Sequence↗
PubMed2006

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 approximately 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.

Amino Acid Sequence↗
PubMed2005

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection.

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.

Codon, Initiator↗
PubMed2005

Pi release from eIF2, not GTP hydrolysis, is the step controlled by start-site selection during eukaryotic translation initiation.

Irreversible GTP hydrolysis by eIF2 is a critical step in translation initiation in eukaryotes because it is thought to commit the translational machinery to assembling the ribosomal complex at the selected point in the mRNA. Our quantitative analysis of the steps and interactions involved in activating GTP hydrolysis by eIF2 during translation initiation in vitro indicates that a structural rearrangement in the 43S preinitiation complex activates it to become fully competent to hydrolyze GTP. Contrary to the prevailing model, release of inorganic phosphate after GTP hydrolysis by eIF2, not hydrolysis itself, is controlled by recognition of the AUG codon. Release of P(i), which makes GTP hydrolysis irreversible, appears to be controlled by the AUG-dependent dissociation of eIF1 from the preinitiation complex.

Codon, Initiator↗
PubMed2005

The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo.

Translation initiation factor 1A stimulates 40S-binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met-tRNA(iMet) ternary complex (TC) and promotes scanning in vitro. eIF1A contains an OB-fold present in bacterial IF1 plus N- and C-terminal extensions. Truncating the C-terminus (deltaC) or mutating OB-fold residues (66-70) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd- phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S-bound TC, eIF5 and eIF3 in vivo, and deltaC impaired TC recruitment in vitro. The assembly defects of the OB-fold mutation can be attributed to reduced 40S-binding of eIF1A, whereas deltaC impairs eIF1A function on the ribosome. A substitution in the C-terminal helix (98-101) also reduced 43S assembly in vivo. Rather than producing a Gcd- phenotype, however, 98-101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98-101 allows formation of aberrant 48S complexes in vitro and increases utilization of non-AUG codons in vivo. Thus, the OB-fold is crucial for ribosome-binding and the C-terminal domain of eIF1A has eukaryotic-specific functions in TC recruitment and scanning.

Codon, Initiator↗
PubMed2005

A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon.

During eukaryotic translation initiation, ribosomal 43S complexes scan mRNAs for the correct AUG codon at which to begin translation. Start codon recognition triggers GTP hydrolysis, committing the complex to engagement at that point on the mRNA. While fidelity at this step is essential, the nature of the codon recognition event and the mechanism by which it activates GTP hydrolysis are poorly understood. Here we report the changes that occur within the 43S.mRNA complex in response to AUG codon recognition. eIF1 and eIF1A are key players in assembly of 43S.mRNA complexes capable of locating initiation codons. We observed FRET between these two factors when bound to the 40S subunit. Using steady-state FRET, anisotropy, and kinetic analyses, we demonstrate that start codon recognition results in a conformational change and release of eIF1 from the ribosome. These rearrangements probably play a role in triggering GTP hydrolysis and committing the complex to downstream events.

Codon, Initiator↗
PubMed2005

Ribozyme catalysis: not different, just worse.

Evolution has resoundingly favored protein enzymes over RNA-based catalysts, yet ribozymes occupy important niches in modern cell biology that include the starring role in catalysis of protein synthesis on the ribosome. Recent results from structural and biochemical studies show that natural ribozymes use an impressive range of catalytic mechanisms, beyond metalloenzyme chemistry and analogous to more chemically diverse protein enzymes. These findings make it increasingly possible to compare details of RNA- and protein-based catalysis.

Animals↗
PubMed2004

GTP-dependent recognition of the methionine moiety on initiator tRNA by translation factor eIF2.

Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis. In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNA(i)) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon. We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2.GTP.Met-tRNA(i) ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2's affinity for Met-tRNA(i) in solution. In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNA(i) that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact. This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway. The toggling on and off of the factor's interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition. In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2. Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2.

Base Pairing↗
PubMed2004

The molecular mechanics of eukaryotic translation.

Great advances have been made in the past three decades in understanding the molecular mechanics underlying protein synthesis in bacteria, but our understanding of the corresponding events in eukaryotic organisms is only beginning to catch up. In this review we describe the current state of our knowledge and ignorance of the molecular mechanics underlying eukaryotic translation. We discuss the mechanisms conserved across the three kingdoms of life as well as the important divergences that have taken place in the pathway.

Archaeal Proteins↗
PubMed2003

Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit.

We have used expressed protein ligation to site-specifically label eukaryotic translation initiation factors (eIFs) 1 and 1A at their C termini with tetramethyl rhodamine. These fluorescent proteins were used in steady-state anisotropy-based binding experiments to measure the dissociation constants of the factors and the yeast small (40S) ribosomal subunit for the first time. These studies demonstrate that both eIF1 and eIF1A are capable of binding to the 40S subunit in the absence of any other initiation factors or mRNA, arguing against previous suggestions that eIF3 is required for recruitment of eIF1 to the small ribosomal subunit. Strikingly, the data also demonstrate that there is approximately ninefold thermodynamic coupling in the binding of the two factors to the 40S subunit. This indicates that eIF1 and eIF1A communicate with one another when bound to the 40S subunit. Communication between these two factors is likely to be important for coordinating their functions during the initiation process. The data presented here provide a foundation on which to build a quantitative understanding of the network of interactions between these essential factors and the rest of the initiation machinery.

Anisotropy↗
PubMed2002

Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity.

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.

Alleles↗
PubMed2002

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.

Eukaryotic Cells↗