scholarly journals Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

1991 ◽  
Vol 11 (4) ◽  
pp. 2149-2153 ◽  
Author(s):  
Y Feng ◽  
L E Gunter ◽  
E L Organ ◽  
D R Cavener
Keyword(s):  
Translation Initiation ◽  
Larval Stage ◽  
Developmental Stages ◽  
Germ Line ◽  
Adult Stage ◽  
Mutant Gene ◽  
Start Codon ◽  
Fold Reduction

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

scholarly journals Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon

1991 ◽  
Vol 11 (4) ◽  
pp. 2149-2153
Author(s):  
Y Feng ◽  
L E Gunter ◽  
E L Organ ◽  
D R Cavener
Keyword(s):  
Translation Initiation ◽  
Larval Stage ◽  
Developmental Stages ◽  
Germ Line ◽  
Adult Stage ◽  
Mutant Gene ◽  
Start Codon ◽  
Fold Reduction

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

scholarly journals eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex

2020 ◽  
Vol 48 (18) ◽  
pp. 10280-10296
Author(s):  
Anil Thakur ◽  
Swati Gaikwad ◽  
Anil K Vijjamarri ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Preinitiation Complex ◽  
Closed Conformation ◽  
Open Conformation ◽  
Kozak Context ◽  
Codon Selection

Abstract In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the −3 nucleotide of the ‘Kozak’ context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui− cells (Ssu− phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui− phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd− phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

scholarly journals The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jyothsna Visweswaraiah ◽  
Yvette Pittman ◽  
Thomas E Dever ◽  
Alan G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Ternary Complex ◽  
Start Codon ◽  
Exit Channel ◽  
Hairpin Loop ◽  
Leaky Scanning ◽  
Initiation Factors ◽  
Codon Recognition

The eukaryotic 43S pre-initiation complex bearing tRNAiMet scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred ‘leaky-scanning’ of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the ‘PIN’ conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.

scholarly journals Identification and Characterization of Functionally Critical, Conserved Motifs in the Internal Repeats and N-terminal Domain of Yeast Translation Initiation Factor 4B (yeIF4B)

2013 ◽  
Vol 289 (3) ◽  
pp. 1704-1722 ◽  
Author(s):  
Fujun Zhou ◽  
Sarah E. Walker ◽  
Sarah F. Mitchell ◽  
Jon R. Lorsch ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Rna Recognition Motif ◽  
Conserved Motifs ◽  
Terminal Domain ◽  
Sequence Elements

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.

scholarly journals Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2

2000 ◽  
Vol 20 (19) ◽  
pp. 7183-7191 ◽  
Author(s):  
Sang Ki Choi ◽  
DeAnne S. Olsen ◽  
Antonina Roll-Mecak ◽  
Agnes Martung ◽  
Keith L. Remo ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Sequence Similarity ◽  
Start Codon ◽  
Amino Acid Sequence Similarity ◽  
Physical Interaction ◽  
Binding Assays ◽  
Initiation Factors ◽  
Translation Initiation Factors

ABSTRACT To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

scholarly journals Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in Escherichia coli

2006 ◽  
Vol 189 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Jay E. Brock ◽  
Robert L. Paz ◽  
Patrick Cottle ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Start Codon ◽  
Coding Sequences ◽  
E Coli ◽  
Ribosome Binding ◽  
Naturally Occurring ◽  
In Vivo Expression

ABSTRACT Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

COVID-19: Opinion in Prevention and dietary based management hypothesis

Coronaviruses ◽  
2020 ◽  
Vol 01 ◽  
Author(s):  
Ashraf Talaat Youssef
Keyword(s):  
Fatty Acids ◽  
Saturated Fatty Acids ◽  
Lung Damage ◽  
Gastric Aspirate ◽  
Enveloped Viruses ◽  
Multiple Organs ◽  
Fold Reduction ◽  
Antiviral Activities

The pandemic of COVID-19 had started in Wuhan city china in late 2019 with a subsequent worldwide spread. The viral infection can seriousely affect multiple organs mainly lungs, kidneys, heart, liver and brain and may lead to respiratory, renal, cardiac or hepatic failure.Vascular thrombosis of unexplained mechanism that may lead to widespread blood clots in multiple organs and cytokine storms that result of overstimulation of the immune system subsequent of lung damage may lead to sudden decompensation due to hypotension and more damage to liver, kidney, brain or lungs.Until now no drug had proved efficient in getting rid of the problem and controlling the pandemic mainly depends on preventive measures.Many preventive measures can be considered to prevent the worldwide spread of viral transmission. Polyunsaturated long chain fatty acids (PUFAs) and the medium chain saturated fatty acids (MCSFAs) and their corresponding monoglycerides had high antiviral activities against the enveloped viruses which reach to more than 10,000 -fold reduction in the viral titres in vitro and in vivo after testing of its gastric aspirate, and can contribute to the systemic immunity against the enveloped viruses.

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

scholarly journals Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

scholarly journals Transcriptional Regulation of PEN-2, a Key Component of the γ-Secretase Complex, by CREB

2006 ◽  
Vol 26 (4) ◽  
pp. 1347-1354 ◽  
Author(s):  
Ruishan Wang ◽  
Yun-wu Zhang ◽  
Ping Sun ◽  
Runzhong Liu ◽  
Xian Zhang ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activity ◽  
Start Codon ◽  
Gamma Secretase ◽  
Mobility Shift ◽  
Endoproteolytic Cleavage ◽  
Notch Cleavage ◽  
Translational Start

ABSTRACT Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's β-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the γ-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPα and Aβ without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

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