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Alan G Hinnebusch - One of the best experts on this subject based on the ideXlab platform.
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The Scanning Mechanism of Eukaryotic Translation Initiation
Annual Review of Biochemistry, 2014Co-Authors: Alan G HinnebuschAbstract:In eukaryotes, the translation Initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic Initiation Factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preInitiation complex (PIC) joins the 5′ end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate rele...
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β hairpin loop of eukaryotic Initiation Factor 1 eif1 mediates 40 s ribosome binding to regulate initiator trnamet recruitment and accuracy of aug selection in vivo
Journal of Biological Chemistry, 2013Co-Authors: Pilar Martinmarcos, Jagpreet S Nanda, Rafael E Luna, Gerhard Wagner, Jon R Lorsch, Alan G HinnebuschAbstract:Recognition of the translation Initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased Initiation at UUG codons (Sui− phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17–21) known to impede eIF1 dissociation in vitro. The eIF1 Sui− mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd− phenotype is likewise suppressed by eIF1 overexpression or the 17–21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preInitiation complex.
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Functional elements in Initiation Factors 1, 1A, and 2β discriminate against poor AUG context and non-AUG start codons.
Molecular and Cellular Biology, 2011Co-Authors: Pilar Martin-marcos, Yuen-nei Cheung, Alan G HinnebuschAbstract:Yeast eIF1 inhibits Initiation at non-AUG triplets, but it was unknown whether it also discriminates against AUGs in suboptimal context. As in other eukaryotes, the yeast gene encoding eIF1 (SUI1) contains an AUG in poor context, which could underlie translational autoregulation. Previously, eIF1 mutations were identified that increase Initiation at UUG codons (Sui− phenotype), and we obtained mutations with the opposite phenotype of suppressing UUG Initiation (Ssu− phenotype). Remarkably, Sui− mutations in eukaryotic translation Initiation Factor 1 (eIF1), eIF1A, and eIF2β all increase SUI1 expression in a manner diminished by introducing the optimal context at the SUI1 AUG, whereas Ssu− mutations in eIF1 and eIF1A decrease SUI1 expression with the native, but not optimal, context present. Therefore, discrimination against weak context depends on specific residues in eIFs 1, 1A, and 2β that also impede selection of non-AUGs, suggesting that context nucleotides and AUG act coordinately to stabilize the preInitiation complex. Although eIF1 autoregulates by discriminating against poor context in yeast and mammals, this mechanism does not prevent eIF1 overproduction in yeast, accounting for the hyperaccuracy phenotype afforded by SUI1 overexpression.
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Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes
Microbiology and Molecular Biology Reviews, 2011Co-Authors: Alan G HinnebuschAbstract:Summary: The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNAiMet) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of Initiation Factors, notably eukaryotic Initiation Factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNAiMet bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNAiMet more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.
Leif A Isaksson - One of the best experts on this subject based on the ideXlab platform.
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mutations in 16s rrna that suppress cold sensitive Initiation Factor 1 affect ribosomal subunit association
FEBS Journal, 2011Co-Authors: Jaroslav M Belotserkovsky, Eric R Dabbs, Leif A IsakssonAbstract:A mutation in the infA gene encoding Initiation Factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site s ...
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Suppression of a cold‐sensitive mutant Initiation Factor 1 by alterations in the 23S rRNA maturation region
FEBS Journal, 2011Co-Authors: Jaroslav M Belotserkovsky, Georgina Isak, Leif A IsakssonAbstract:Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive Initiation Factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map t ...
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Translation Initiation region dependency of translation Initiation in Escherichia coli by IF1 and kasugamycin
FEBS Journal, 2010Co-Authors: Serhiy Surkov, Hanna Nilsson, Louise Carøe Vohlander Rasmussen, Hans Uffe Sperling-petersen, Leif A IsakssonAbstract:Translation Initiation Factor 1 (IF1) is an essential protein in prokaryotes. The nature of IF1 interactions with the mRNA during translation Initiation on the ribosome remains unclear, even though ...
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A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli
Journal of Biotechnology, 2004Co-Authors: Peter Jörgen Hägg, Johanna Wa De Pohl, Farhad Maruf Abdulkarim, Leif A IsakssonAbstract:Abstract Uneven distribution of plasmid-based expression vectors to daughter cells during bacterial cell division results in an increasing proportion of plasmid free cells during growth. This is a major industrial problem leading to reduction of product yields and increased production costs during large-scale cultivation of vector-carrying bacteria. For this reason, a selection must be provided that kills the plasmid free cells. The most conventional method to obtain this desired selection is to insert some gene for antibiotic resistance in the plasmid and then grow the bacteria in the presence of the corresponding antibiotic. We describe here a host/plasmid Escherichia coli system with a totally stable plasmid that can be maintained without the use of antibiotic selection. The plasmid is maintained, since it carries the small essential gene infA (coding for translation Initiation Factor 1, IF1) in an E. coli strain that has been deleted for its chromosomal infA gene. As a result only plasmid carrying cells can grow, making the strain totally dependent on the maintenance of the plasmid. A selection based on antibiotics is thus not necessary during cultivation, and no antibiotic-resistance genes are present neither in the final strain nor in the final plasmid. Plasmid-free cells do not accumulate even after an extended period of continuous growth. Growth rates of the control and the plasmid harboring strains are indistinguishable from each other in both LB and defined media. The indicated approach can be used to modify existing production strains and plasmids to the described concept. The infA based plasmid stability system should eliminate industrial cultivation problems caused by the loss of expression vector and use of antibiotics in the cultivation medium. Also environmental problems caused by release of antibiotics and antibiotic resistance genes, that potentially can give horizontal gene transfer between bacterial populations, are eliminated.
Shailja Misrabhattacharya - One of the best experts on this subject based on the ideXlab platform.
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functional attributes of evolutionary conserved arg45 of wolbachia brugia malayi translation Initiation Factor 1
Future Microbiology, 2016Co-Authors: Dhanvantri Chahar, Nidhi Shrivastava, Chhedi Lal Gupta, Preeti Bajpai, Deepak Chandra, Shailja MisrabhattacharyaAbstract:Aim: Wolbachia is a promising antifilarial chemotherapeutic target. Translation Initiation Factor-1 (Tl IF-1) is an essential Factor in prokaryotes. Functional characterization of Wolbachia’s novel proteins/enzymes is necessary for the development of adulticidal drugs. Materials & methods: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. Results: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. Conclusion: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.
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recombinant translation Initiation Factor 1 of wolbachia is an immunogenic excretory secretory protein that elicits th2 mediated immune protection against brugia malayi
Comparative Immunology Microbiology and Infectious Diseases, 2013Co-Authors: Nidhi Shrivastava, Jyoti Gupta, Shailja MisrabhattacharyaAbstract:Wolbachia, the intracellular alpha-proteobacteria are required for the development, fertility and survival of filarial parasites. Wolbachia Translation Initiation Factor-1 (Wol Tl IF-1) is one of the Factors required for Wolbachia growth and viability. In the present study, we cloned, over expressed and purified Wol Tl IF-1 that exhibited strong immuno-reactivity with various categories of bancroftian sera. Immunization with the recombinant protein resulted into significant reduction in microfilarial density (70–72%) and adult worm establishment (61–63%) in susceptible Mastomys coucha. Protection offered by Wol Tl IF-1 was found associated with humoral immune arm as observed by an increased antibody level with preponderance of IgE, IgM, IgG1 and IgG2a isotypes. The anti-Wol Tl IF-1 antibodies promoted profound adherence of peritoneal exudates cells to the surface of microfilariae and infective larvae causing cytotoxicity and their death. The present study indicates potential of recombinant Wol Tl IF-1 as a promising vaccine candidate against human lymphatic filarial infection.
Yonghong Zhang - One of the best experts on this subject based on the ideXlab platform.
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1 h 13 c and 15 n resonance assignments and structure prediction of translation Initiation Factor 1 from clostridium difficile
Biomolecular Nmr Assignments, 2019Co-Authors: Faith Aguilar, Niaz Banaei, Yonghong ZhangAbstract:Clostridium difficile is a gram-positive, toxin-producing, anaerobic bacterium whose virulence Factors and mechanisms of pathogenesis require further investigation. C. difficile infections (CDI) result in the severe and potentially fatal gastrointestinal diseases pseudomembranous colitis and toxic megacolon following extensive broad spectrum antibiotic treatment. The increasing C. difficile fatalities are a result of the bacteria’s growing antibiotic resistance and consequential CDI recurrence, which led to the unmet need for new CDI treatment. Bacterial protein synthesis is an essential metabolic process and an effective target for antibacterial agents. Translation Initiation Factor 1 from C. difficile (Cd-IF1) is the smallest of the three Initiation Factors that acts to establish the 30S Initiation complex to initiate translation during protein biosynthesis. Here we report the complete NMR 1H, 13C and 15N chemical shift assignments of Cd-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses have identified five β-strands and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5, which is supported by 15N-{1H} heteroNOEs. The assigned chemical shifts were used to conduct structure prediction by CS-Rosetta. The predicted structure suggests that Cd-IF1 adopts the typical β-barrel structure and is composed of an oligomer-binding motif.
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^1H, ^13C and ^15N resonance assignments and secondary structure analysis of translation Initiation Factor 1 from Pseudomonas aeruginosa
Biomolecular NMR Assignments, 2016Co-Authors: Alejandra Bernal, Yanmei Hu, James M. Bullard, Stephanie O. Palmer, Aaron Silva, Yonghong ZhangAbstract:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation Initiation Factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three Initiation Factors that acts to establish the 30S Initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the ^1H, ^13C and ^15N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five β-strands with an unusually extended β-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order β1–β2–β3–α1–β4–β5. This is further supported by ^15N–{^1H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical β-barrel structure and is composed of an oligomer-binding motif.
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Solution structure of protein synthesis Initiation Factor 1 from Pseudomonas aeruginosa
Protein Science, 2016Co-Authors: Yanmei Hu, Alejandra Bernal, James M. Bullard, Yonghong ZhangAbstract:Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation Factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three Initiation Factors that act to establish the 30S Initiation complex during Initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa-IF1. Pa-IF1 consists of a five-stranded β-sheet with an unusual extended β-strand at the C-terminus and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5. The structure adopts a typical β-barrel fold and contains an oligomer-binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α-helix may compose the binding interface with the 30S subunit.
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(1)H, (13)C and (15)N resonance assignments and secondary structure analysis of translation Initiation Factor 1 from Pseudomonas aeruginosa.
Biomolecular Nmr Assignments, 2016Co-Authors: Alejandra Bernal, Yanmei Hu, James M. Bullard, Stephanie O. Palmer, Aaron Silva, Yonghong ZhangAbstract:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation Initiation Factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three Initiation Factors that acts to establish the 30S Initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the 1H, 13C and 15N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five β-strands with an unusually extended β-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order β1–β2–β3–α1–β4–β5. This is further supported by 15N–{1H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical β-barrel structure and is composed of an oligomer-binding motif.
Albert J Fornace - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of a human genotoxic and endoplasmic reticulum stress inducible cdna that encodes translation Initiation Factor 1 eif1 a121 sui1
Journal of Biological Chemistry, 1999Co-Authors: Saeed M Sheikh, Ester Fernandezsalas, Myounghee Yu, Arif Hussain, Jonathan D Dinman, Stuart W Peltz, Ying Huang, Albert J FornaceAbstract:Abstract We report the cloning and characterization of a DNA damage-inducible (DDI) transcript DDI A121. The full-length human DDI A121 cDNA contains an open reading frame of 113 amino acids, corresponding to a protein of 12.7 kDa. The deduced amino acid sequence of A121 shows high homology to the yeast translation Initiation Factor (eIF) sui1 and also exhibits perfect identity to the partial sequence of recently purified human eIF1. Expression of human A121 corrected the mutant sui1 phenotype in yeast, demonstrating that human A121 encodes abona fide translation Initiation Factor that is equivalent to yeast sui1p. The mammalian A121/SUI1 gene exhibits two transcripts (1.35 kilobases and 0.65 kilobases) containing a common coding region but differing in their 3′-untranslated region. The long and short A121/SUI1 mRNAs are differentially regulated by genotoxic and endoplasmic reticulum stress. The genotoxic stress induction of A121/SUI1 mRNA is conserved in both humans and rodents and occurs in a p53-independent manner. Our identification of a stress-inducible cDNA that encodes eIF1 suggests that modulation of translation Initiation appears to occur during cellular stress and may represent an important adaptive response to genotoxic as well as endoplasmic reticulum stress.