Sarah E. Walker

Multiple elements in the eIF4G1 N‐terminus promote assembly of eIF4G1•PABP mRNPs in vivo

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)‐binding protein (PABP) are thought to enhance formation of activated eIF4F•mRNA•PABP complexes competent to recruit 43S pre‐initiation complexes. We found that the RNA‐binding region (RNA1) in the N‐terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP‐binding segment to rescue the function of an eIF4G1‐459 mutant impaired for eIF4E binding. Assaying RNA‐dependent PABP–eIF4G association in cell extracts suggests that RNA1, the PABP‐binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4G•mRNA•PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G–mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP–eIF4G association is only one of several interactions that stabilize eIF4F•mRNA complexes, and emphasize that closed‐loop mRNP formation via PABP–eIF4G interaction is non‐essential in vivo. Interestingly, two other RNA‐binding regions in eIF4G1 have critical functions downstream of eIF4F•mRNA assembly.

Initiation factor eIF2γ promotes eIF2–GTP–Met-tRNAiMet ternary complex binding to the 40S ribosome

In contrast to prokaryotic elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the ribosomal A-site, eukaryotic initiation factor eIF2 binds methionyl initiator transfer RNA (Met-tRNAiMet) to the P-site of the 40S ribosomal subunit. The results of directed hydroxyl radical probing experiments to map binding of Saccharomyces cerevisiae eIF2 on the ribosome and on Met-tRNAiMet revealed that eIF2γ primarily contacts the acceptor stem of Met-tRNAiMet and identified a key binding interface between domain III of eIF2γ and 18S rRNA helix h44 on the 40S subunit. Whereas the analogous domain III of EF-Tu contacts the T stem of tRNAs, biochemical analyses demonstrated that eIF2γ domain III is important for ribosome, not Met-tRNAiMet. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new ribosome-binding function in eIF2γ.

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.

Yeast Eukaryotic Initiation Factor 4B (eIF4B) Enhances Complex Assembly between eIF4A and eIF4G in Vivo

Background: Mammalian eIF4B stimulates eIF4A helicase activity, but its function in promoting translation initiation in yeast is unclear. Results: Yeast eIF4B enhances eIF4G·eIF4A association in vivo and in vitro. Conclusion: yeIF4B stimulates eIF4F assembly by promoting an eIF4G HEAT domain conformation conducive for binding eIF4A. Significance: A new function is established for eIF4B of supporting eIF4F assembly for mRNA activation. Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m7G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts− mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

RNA purification - Precipitation methods

When working with RNA, the need often arises to concentrate a sample or purify it from various salts, nucleotides, and proteins. RNA precipitation is an easy and cost-effective method for the concentration of RNA, leaving a pellet that can be resuspended in the buffer of choice.

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.

Sanger dideoxy sequencing of DNA.

While the ease and reduced cost of automated DNA sequencing has largely obviated the need for manual dideoxy sequencing for routine purposes, specific applications require manual DNA sequencing. For instance, in studies of enzymes or proteins that bind or modify DNA, a DNA ladder is often used to map the site at which an enzyme is bound or a modification occurs. In these cases, the Sanger method for dideoxy sequencing provides a rapid and facile method for producing a labeled DNA ladder.

Chapter Nineteen – RNA Purification – Precipitation Methods

When working with RNA, the need often arises to concentrate a sample or purify it from various salts, nucleotides, and proteins. RNA precipitation is an easy and cost-effective method for the concentration of RNA, leaving a pellet that can be resuspended in the buffer of choice.

Sanger dideoxy sequencing of DNA

While the ease and reduced cost of automated DNA sequencing has largely obviated the need for manual dideoxy sequencing for routine purposes, specific applications require manual DNA sequencing. For instance, in studies of enzymes or proteins that bind or modify DNA, a DNA ladder is often used to map the site at which an enzyme is bound or a modification occurs. In these cases, the Sanger method for dideoxy sequencing provides a rapid and facile method for producing a labeled DNA ladder.

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.