Katsura Asano

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.

Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site.

Replication of a low‐copy‐number IncIα plasmid ColIb‐P9 depends on expression of the repZ gene encoding the replication initiator protein. repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem–loop I, the Inc RNA target, and a downstream sequence complementary to the loop I. The loop I sequence comprises 5′‐rUUGGCG‐3′, conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control. Here we show that the level of repZ expression is rate‐limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb‐P9 derivatives defective in Inc RNA and pseudoknot formation. Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I. This initial interaction is stimulated by the presence of the loop U residue in the 5′‐rUUGGCG‐3′ motif. These results indicate that the competition between the two RNA–RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.

Study of Translational Control of Eukaryotic Gene Expression Using Yeast

Abstract: Eukaryotic cells respond to starvation by decreasing the rate of general protein synthesis while inducing translation of specific mRNAs encoding transcription factors GCN4 (yeast) or ATF4 (humans). Both responses are elicited by phosphorylation of translation initiation factor 2 (eIF2) and the attendant inhibition of its nucleotide exchange factor eIF2B—decreasing the binding to 40S ribosomes of methionyl initiator tRNA in the ternary complex (TC) with eIF2 and GTP. The reduction in TC levels enables scanning ribosomes to bypass the start codons of upstream open reading frames in the GCN4 mRNA leader and initiate translation at the authentic GCN4 start codon. We exploited the fact that GCN4 translation is a sensitive reporter of defects in TC recruitment to identify the catalytic and regulatory subunits of eIF2B. More recently, we implicated the C‐terminal domain of eIF1A in 40S‐binding of TC in vivo. Interestingly, we found that TC resides in a multifactor complex (MFC) with eIF3, eIF1, and the GTPase‐activating protein for eIF2, known as eIF5. Our biochemical and genetic analyses indicate that physical interactions between MFC componens enhance TC binding to 40S subunits and are required for wild‐type translational control of GCN4. MFC integrity and eIF3 function also contribute to post‐assembly steps in the initiation pathway that impact GCN4 expression. Thus, apart from its critical role in the starvation response, GCN4 regulation is a valuable tool for dissecting the contributions of multiple translation factors in the eukaryotic initiation pathway.