Sarah F. Mitchell

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.

N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection

Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C‐terminal tail (CTT) of eIF1A impairs recruitment of the eIF2‐GTP‐Met‐tRNAiMet ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17–21 in the N‐terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui− phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17–21 suppresses the Sui− phenotypes of eIF5 and eIF2β mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning‐conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

Start codon selection is a key step in translation initiation as it sets the reading frame for decoding. Two eukaryotic initiation factors, eIF1 and eIF1A, are key actors in this process. Recent work has elucidated many details of the mechanisms these factors use to control start site selection. eIF1 prevents the irreversible GTP hydrolysis that commits the ribosome to initiation at a particular codon. eIF1A both promotes and inhibits commitment through the competing influences of its two unstructured termini. Both factors perform their tasks through a variety of interactions with other components of the initiation machinery, in many cases mediated by the unstructured regions of the two proteins.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4Bs domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosomes mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

Ribosome recycling step in yeast cytoplasmic protein synthesis is catalyzed by eEF3 and ATP.

After each round of protein biosynthesis, the posttermination complex (PoTC) consisting of a ribosome, mRNA, and tRNA must be disassembled into its components for a new round of translation. Here, we show that a Saccharomyces cerevisiae model PoTC was disassembled by ATP and eukaryotic elongation factor 3 (eEF3). GTP or ITP functioned with less efficiency and adenosine 5γ′-(β,γ-imido)triphosphate did not function at all. The kcat of eEF3 was 1.12 min-1, which is comparable to that of the in vitro initiation step. The disassembly reaction was inhibited by aminoglycosides and cycloheximide. The subunits formed from the yeast model PoTC remained separated under ionic conditions close to those existing in vivo, suggesting that they are ready to enter the initiation process. Based on our experimental techniques used in this paper, the release of mRNA and tRNA and ribosome dissociation took place simultaneously. No 40S•mRNA complex was observed, indicating that eEF3 action promotes ribosome recycling, not reinitiation.

Identification and characterization of functionally critical, conserved motifs in the internal repeats and N-terminal domain of yeast translation initiation factor 4B (yeIF4B).

Background: The internal repeats and NTD of yeIF4B stimulate translation initiation. Results: The minimal number of repeats and conserved motifs in the repeat and NTD necessary for yeIF4B function was determined. Conclusion: Two repeats provide appreciable function, except when the NTD is missing or eIF4F function is limiting or compromised. Significance: The results provide a comprehensive description of functionally critical sequence elements in yeIF4B. eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

Standard In Vitro Assays for Protein–Nucleic Acid Interactions – Gel Shift Assays for RNA and DNA Binding

The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed.

Protein Affinity Purification using Intein/Chitin Binding Protein Tags

Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protein of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.

Recruiting knotty partners: The roles of translation initiation factors in mRNA recruitment to the eukaryotic ribosome

Eukaryotic translation initiation begins with the binding of a ternary complex (TC) composed of eukaryotic initiation factor (eIF) 2, methionyl initiator tRNA (Met-tRNAi) and GTP to the 40S ribosomal subunit to form the 43S pre-initiation complex (PIC) in a process that is promoted by eIFs 1, 1A, and 3 (Figure 1; for reviews of the entire pathway of translation initiation see (Jackson et al., 2010; Lorsch and Dever, 2010)). This complex is then capable of binding the mRNA near the 5′ end and moving in a 5′-to-3′ direction in search of the start codon. As the start codon in eukaryotic mRNA is usually the first AUG codon, it is necessary for the ribosome to be recruited to the very 5′ end of the mRNA. Were it to bind 3′ to the start codon, it would scan to the next AUG and make an aberrant polypeptide. This localization is achieved by the presence of a 7-methylguanosine cap on the 5′ end of the mRNA that, through a number of protein-protein interactions, brings the ribosome to the very beginning of the mRNA. Protein factors are also thought to be responsible for removing secondary structure and RNA binding proteins from the RNA to create a single-stranded region for the ribosome to bind. Interaction between factors at the 5′ and 3′ ends of the message functionally circularizes the mRNA and allows communication between the ends. After initial recruitment of the PIC, many of these factors are also thought to be involved in the process of scanning, removing structure and proteins so that the ribosome can move forward in search of the start codon, and potentially also pushing the PIC along the mRNA or increasing the directionality of this movement.

Protein Derivitization-Expressed Protein Ligation

Expressed protein ligation (EPL) combines two methods to ligate a synthetic peptide to a recombinant protein. Native chemical ligation (NCL) is a process in which two synthesized peptides are ligated by reaction of a C-terminal thioester on one peptide with an N-terminal cysteine residue of another protein. The chemistry of inteins, self-excising protein fragments that ligate the surrounding protein back together, creates isolatable intermediates with the two chemical groups necessary for NCL, a C-terminal thioester and an N-terminal cysteine residue. This technique allows for the incorporation of synthetic amino acids, radiolabeled amino acids, and fluorescent moieties at specific locations in a protein. It has the advantage of allowing attachment of such synthetic peptides to the termini of a recombinant protein, allowing for the synthesis of large proteins with modified amino acids. This technique utilizes the IMPACT(TM)-System created by New England Biolabs, who provide a variety of vectors in which the multicloning site is directly upstream of an intein sequence fused to a chitin-binding domain (CBD). The CBD binds tightly and specifically to chitin beads, allowing for an efficient one-step purification. This step can be used to obtain highly purified proteins (see Protein Affinity Purification using Intein/Chitin Binding Protein Tags). After purification of the recombinant protein, cleavage from the intein is achieved through the addition of a reactive thiol compound, usually sodium 2-mercaptoethanesulfonate (MESNA) (see also Proteolytic affinity tag cleavage). This reaction creates a protein with a C-terminal thioester that can then react with a peptide containing an N-terminal cysteine residue, ligating the two proteins via a peptide bond.