Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast

The eukaryotic translation elongation factor 2 (eEF2), a member of the G-protein superfamily, catalyzes the post-peptidyl transferase translocation of deacylated tRNA and peptidyl tRNA to the ribosomal E- and P-sites. eEF2 is modified by a unique post-translational modification: the conversion of His699 to diphthamide at the tip of domain IV, the region proposed to mimic the anticodon of tRNA. Structural models indicate a hinge is important for conformational changes in eEF2. Mutations of V488 in the hinge region and H699 in the tip of domain IV produce non-functional mutants that when co-expressed with the wild-type eEF2 result in a dominant-negative growth phenotype in the yeast Saccharomyces cerevisiae. This phenotype is linked to reduced levels of the wild-type protein, as total eEF2 levels are unchanged. Changes in the promoter, 5′-untranslated region (5′-UTR) or 3′-UTR of the EFT2 gene encoding eEF2 do not allow overexpression of the protein, showing that eEF2 levels are tightly regulated. The H699K mutant, however, also alters translation phenotypes. The observed regulation suggests that the cell needs an optimum amount of active eEF2 to grow properly. This provides information about a new mechanism by which translation is efficiently maintained

Redbird Buzz Episode 1: Terri Goss Kinzy, May 2022

Interview with Illinois State University\u27s 20th president, Terri Goss Kinzy. The interview was conducted in May 2022 by Rachel Kobus from Alumni Engagement, for the Illinois State University Redbird Buzz Podcast

Public Impact-Focused Research Survey Results

The Association of Public and Land Grant Universities (APLU) Council on Research (COR) led an initiative to define, identify, and develop a recommended path forward for public impact research (PIR). A survey was conducted of APLU institution in order to: To characterize the extent of public impact research (PIR) occurring at APLU institutions. To understand how institutions (or leaders within institutions) think about, define, and communicate about this type of work. To provide perspectives about the challenges, opportunities, and rewards that may be associated with this type of scholarship. Responses were received from a diverse set of seventy public and land grant universities (APLU total membership was 239 universities at the time of this survey). Research expenditures at responding institutions ranged from $5 million to over$1 billion in FY 2017, and respondents included Hispanic-serving institutions, historically black universities, IEP-designated universities, and were received from 26 US states and one Canadian province. This document contains the complete set of de-identified responses to the survey. The intent is to make this broadly available and accessible to individuals or groups who may want to further analyze or use these results

Characterization of GTP and Aminoacyl-tRNA Binding to Eukaryotic Initiation Factor 2 and Elongation Factor 1

Eukaryotic protein synthesis requires two protein factors which bind aminoacyl-tRNA in a GTP-dependent manner. One, initiation factor 2 (eIF-2), binds only the initiator Met-tRNAi and carries it to the 40S subunit of the ribosome. The other, elongation factor 1α (EF-1α) binds all other aminoacyl-tRNAs to the elongating 80S ribosome. The goal of this thesis is to characterize the structural aspects of these proteins

Synchronicity: Policing Multiple Aspects of Gene Expression by Ctk1

Transcription and translation are coordinated events in all organisms. In prokaryotes, the process that couples these two events is clear: The ribosome begins translation of the nascent mRNA while the DNA template is still being transcribed. Indeed, cotranscriptional protein synthesis underlies key regulatory mechanisms in bacteria, including attenuation, the mechanism that regulates RNA polymerase processivity in response to ribosome movement along the mRNA. But how is transcription coordinated with translation in eukaryotic organisms, where mRNA is synthesized in the nucleus and protein synthesis occurs in the cytoplasm? Although these two events are spatially distinct, separated by the nuclear envelope, efficient control of gene expression necessarily requires that transcription and translation be regulated in a coordinated manner. As an example, dFOXO-mediated transcriptional activation produces both an inhibitor of cap-dependent translation, eukaryotic translation initiation factor 4E (eIF4E)-BP, and a form of the insulin receptor mRNA that is translated by a cap-independent mechanism (Marr et al. 2007). In addition, translation requires a fully and accurately processed mRNA, and has mechanisms to help sense that appropriate processing has occurred

Nontranslational Functions of Components of the Translational Apparatus

A growing number of studies have identified new and often nontranslational functions for many components of the translational apparatus, including tRNAs, aminoacyl-tRNA synthetases, ribosomal proteins, and initiation and elongation factors (Fig. 1). Although initially the sheer abundance of some of these components made their identification in novel functions suspect, increasingly detailed biochemical and genetic studies have established the multifunctional nature of these molecules. The studies include classic and elegant examples of viral systems that recruit host factors for their replication and maintenance, as well as cellular processes that adapt these abundant cellular components to new and perhaps related functions. Furthermore, the increasing recognition that some translational components reside in previously unexpected locations may serve to link the regulation of gene expression and the quality of protein synthesis with new functions of the translational apparatus itself (Fig. 1)

Yeast as a Sensor of Factors Affecting the Accuracy of Protein Synthesis

The cell monitors and maintains the fidelity of translation during the three stages of protein synthesis: initiation, elongation and termination. Errors can arise by multiple mechanisms, such as altered start site selection, reading frame shifts, misincorporation or nonsense codon suppression. All of these events produce incorrect protein products. Translational accuracy is affected by both cis- and trans-acting elements that insure the proper peptide is synthesized by the protein synthetic machinery. Many cellular components are involved in the accuracy of translation, including RNAs (transfer RNAs, messenger RNAs and ribosomal RNAs) and proteins (ribosomal proteins and translation factors). The yeast Saccharomyces cerevisiae has proven an ideal system to study translational fidelity by integrating genetic approaches with biochemical analysis. This review focuses on the ways studies in yeast have contributed to our understanding of the roles translation factors and the ribosome play in assuring the accuracy of protein synthesis

Expanding the Ribosomal Universe

In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery

Mutations in the Chromodomain-like Insertion of Translation Elongation Factor 3 Compromise Protein Synthesis Through Reduced ATPase Activity

Translation elongation is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic elongation factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome, whereas eEF2 (EF-G in bacteria) translocates the ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP binding cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7d region of the chromodomain-like insertion. Specific mutations in this domain result in reduced growth rate as well as slower translation elongation. In vitro analysis demonstrates that these mutations do not affect the ability of eEF3 to interact with the ribosome. Kinetic analysis revealed a larger turnover number for ribosomes in comparison to eEF3, indicating that the partial reactions involving the ribosome are significantly faster than that of eEF3. Mutations in the chromodomain-like insertion severely compromise the ribosome stimulated ATPase of eEF3, strongly suggesting that it exerts an allosteric effect on the hydrolytic activity of eEF3. The chromodomain-like insertion is, therefore, vital to eEF3 function and may be targeted for developing novel antifungal drugs