Umadas Maitra

The Saccharomyces cerevisiae TIF6 Gene Encoding Translation Initiation Factor 6 Is Required for 60S Ribosomal Subunit Biogenesis

ABSTRACT Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. InSaccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

The Saccharomyces cerevisiae homologue of mammalian translation initiation factor 6 does not function as a translation initiation factor

ABSTRACT Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M r 25,550), designated TIF6, has been cloned and expressed inEscherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.

Distinct Functions of Eukaryotic Translation Initiation Factors eIF1A and eIF3 in the Formation of the 40 S Ribosomal Preinitiation Complex

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S·eIF3·Met-tRNAf·eIF2·GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf·eIF2·GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.

Specific interaction of eukaryotic translation initiation factor 5 (eIF5) with the beta-subunit of eIF2.

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S·mRNA· eIF3·Met-tRNAf·eIF2·GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude initiation factor preparations. We observed that eIF5 specifically interacted with the β subunit of initiation factor eIF2. No other initiation factors including the γ subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the β subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2β deletion mutants expressed inEscherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2β. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNAf·eIF2·GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the β subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation.

Function of Eukaryotic Translation Initiation Factor 1A (eIF1A) (Formerly Called eIF-4C) in Initiation of Protein Synthesis

We have used an efficient in vitro translation initiation system to show that the mammalian 17-kDa eukaryotic initiation factor, eIF1A (formerly designated eIF-4C), is essential for transfer of the initiator Met-tRNAf (as Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA to form the 40 S preinitiation complex (40 S·Met-tRNAf·eIF2·GTP). Furthermore, eIF1A acted catalytically in this reaction to mediate highly efficient transfer of the Met-tRNAf·eIF2·GTP ternary complex to 40 S ribosomal subunits. The 40 S complex formed was free of eIF1A indicating that its role in 40 S preinitiation complex formation is not to stabilize the binding of Met-tRNAf to 40 S ribosomes. Additionally, the eIF1A-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. In contrast to other reports, we found that eIF1A plays no role either in the subunit joining reaction or in the generation of ribosomal subunits from 80 S ribosomes. Our results indicate that the major function of eIF1A is to mediate the transfer of Met-tRNAf to 40 S ribosomal subunits to form the 40 S preinitiation complex.

[27] Isolation and properties of protein factors involved in polypeptide chain initiation in Escherichia coli☆

Publisher Summary This chapter describes a method that yields highly purified initiation factors, and free of transfer factor or nucleoside triphosphatase contamination. The initiation of protein synthesis in Escherichia coli proceeds with the formation of a complex consisting of mRNA, a 70 S ribosome, and the specific initiator, formylmethionyl-tRNA (fMet-tRNA). Formation of the initiation complex requires GTP, Mg 2+ , and NH 4+ ions and, in addition, several protein factors that are loosely associated with ribosomes and can be dissociated by high salt treatment. The purification procedure for the initiation factors involves preliminary separation of initiation factors on DEAE-Cellulose, followed by further purification of initiation factors FI, FII, and FIII by various types of chromatography. FI is a heat-stable, basic protein consisting of a single polypeptide chain of molecular weight 9000. FII is also a basic protein, based upon its elution from CM-Sephadex and its migration in gel electrophoresis, and consists of a single polypeptide chain of molecular weight 21,000, while FIII is an acidic, heat-labile protein, and polyacrylamide gel electrophoresis of FIII phosphocellulose fractions reveals 1-4 protein bands, depending upon the position of elution from the phosphocellulose column.

Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo

ABSTRACT Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S–eIF3–AUG–Met-tRNAf–eIF2–GTP) to promote the hydrolysis of ribosome-bound GTP. eIF5 also forms a complex with eIF2 by interacting with the β subunit of eIF2. In this work, we have used a mutational approach to investigate the importance of eIF5-eIF2β interaction in eIF5 function. Binding analyses with recombinant rat eIF5 deletion mutants identified the C terminus of eIF5 as the eIF2β-binding region. Alanine substitution mutagenesis at sites within this region defined several conserved glutamic acid residues in a bipartite motif as critical for eIF5 function. The E346A,E347A and E384A,E385A double-point mutations each caused a severe defect in the binding of eIF5 to eIF2β but not to eIF3-Nip1p, while a eIF5 hexamutant (E345A,E346A,E347A,E384A,E385A,E386A) showed negligible binding to eIF2β. These mutants were also severely defective in eIF5-dependent GTP hydrolysis, in 80S initiation complex formation, and in the ability to stimulate translation of mRNAs in an eIF5-dependent yeast cell-free translation system. Furthermore, unlike wild-type rat eIF5, which can functionally substitute for yeast eIF5 in complementing in vivo a genetic disruption of the chromosomal copy of the TIF5 gene, the eIF5 double-point mutants allowed only slow growth of this ΔTIF5 yeast strain, while the eIF5 hexamutant was unable to support cell growth and viability of this strain. These findings suggest that eIF5-eIF2β interaction plays an essential role in eIF5 function in eukaryotic cells.

Characterization of Multiple mRNAs That Encode Mammalian Translation Initiation Factor 5 (eIF-5)

Eukaryotic translation initiation factor 5 (eIF-5) interacts with the 40 S initiation complex (40S·mRNA·MettRNAf·eIF-2·GTP) to promote the hydrolysis of bound GTP with the concomitant joining of the 60 S ribosomal subunit to the 40 S initiation complex to form a functional 80 S initiation complex. In this paper, the multiple mRNAs that encode mammalian eIF-5 have been characterized. In rat tissues, three major eIF-5 mRNAs of 3.5, 2.8, and 2.2 kilobases in length are detected. All major eIF-5 mRNAs are initiated from a single transcription initiation site, contain identical 5′-untranslated and coding regions, but differ from one another only in the length of their 3′-untranslated regions. The different lengths of the 3′-untranslated region of eIF-5 mRNAs are generated by the use of alternative polyadenylation signals. Additionally, we demonstrate tissue-specific variations in eIF-5 mRNA expression as well as preference for polyadenylation sites. These results should lead to increased understanding of the regulation of eIF-5 gene expression.

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3) MOLECULAR CLONING REVEALS THAT p110 SUBUNIT IS THE MAMMALIAN HOMOLOGUE OF SACCHAROMYCES CEREVISIAE PROTEIN Prt1

Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNAf (as a Met-tRNAf·eIF2·GTP ternary complex) to 40 S ribosomal subunits. Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations. Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction. Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3. The derived amino acid sequence (calculated M r 95,214) shows that the p110 subunit is the mammalian homologue ofSaccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.

Role of DNA in RNA synthesis: XI. Selective transcription of λ DNA segments in vitro by RNA polymerase of Escherichia coli☆☆☆

Abstract In vitro transcription of native DNA isolated from mature bacteriophage λ was studied using highly purified preparations of DNA-dependent RNA polymerase isolated from E. coli W. Half-length segments of sheared λ DNA were separated by density-gradient centrifugation, and the RNA polymerase products synthesized on the whole λ DNA template and on each of its separated halves were characterized with regard to their nearest-neighbor nucleotide frequencies, base composition, average chain-length, sedimentation velocity, and ability to anneal with specific segments of the template. The priming efficiencies of the λ DNA halves were compared, and the influence of certain alterations in the secondary or tertiary structure of the λ DNA template on the RNA products formed in vitro was examined. These studies indicate that transcription of native λ DNA by the E. coli polymerase in vitro is not random; specific template regions present predominantly on the AT-rich (right) half of linear λ DNA are preferentially transcribed throughout the duration of in vitro RNA synthesis. Denaturation of the λ DNA template results in elimination of selective copying. Neither free cohesive ends nor linearly intact DNA are essential for the selection mechanism.