scholarly journals Nonoptimal Codon Usage Is Critical for Protein Structure and Function of the Master General Amino Acid Control Regulator CPC-1

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Xueliang Lyu ◽  
Yi Liu
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Synonymous Codon ◽  
Critical Role ◽  
Amino Acid Starvation ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Control Response ◽  
And Function

ABSTRACT Under amino acid starvation conditions, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control Protein 1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here, we show that Neurospora cpc-1 has a nonoptimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered the CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activation of the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein folding. IMPORTANCE The general amino acid control response is critical for adaptation of organisms to amino acid starvation conditions. The preference to use certain synonymous codons is a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we showed that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, an altered protein degradation rate, and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein structure.

scholarly journals Non-optimal codon usage is critical for protein structure and function of the master general amino acid control regulator CPC-1

2020 ◽  
Author(s):  
Xueliang Lyu ◽  
Yi Liu
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Synonymous Codon ◽  
Critical Role ◽  
Amino Acid Starvation ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Control Response ◽  
And Function

ABSTRACTUnder amino acid starvation condition, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control-1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here we show that Neurospora cpc-1 has a non-optimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activating the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulating of CPC-1 expression and function, and establish a genetic example of the importance of codon usage in protein structure.Abstract importanceGeneral amino acid control response is critical for organisms to adapt to amino acid starvation condition. The preference to use certain synonymous codons are a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we show that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, altered protein degradation rate and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulating of CPC-1 expression and function, and establish a genetic example of the importance of codon usage in protein structure.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

The yeast ILV2 gene is under general amino acid control

Genome ◽  
1988 ◽  
Vol 30 (6) ◽  
pp. 984-986 ◽  
Author(s):  
W. Xiao ◽  
G. H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Key Words ◽  
Acetolactate Synthase ◽  
Amino Acid Starvation ◽  
Sulfometuron Methyl ◽  
General Amino Acid Control ◽  
Acid Control ◽  
High Level ◽  
Level Resistance

The yeast ILV2 gene encodes acetolactate synthase, the first enzyme in the biosynthesis of isoleucine and valine. Its multiple regulation has precluded the clear demonstration of whether ILV2 is under general amino acid control. Nonderepressible gcn4 strains were used as recipients for transformation with a YCp plasmid carrying GCN4. Parental gcn4 cells and their isogenic GCN4 transformants were evaluated for ALS derepression following induced amino acid starvation. GCN4 cells showed 1.5-to 1.7-fold derepression but no derepression was observed in isogenic control gcn4 strains. A similar depression of ILV2 mRNA was also observed. Genetic evidence for general amino acid control was the gcn4 suppression of high level resistance to sulfometuron methyl by the SMR1-410 allele of ILV2.Key words: Saccharomyces cerevisiae, ILV2 gene, general amino acid control, multiple regulators.

scholarly journals The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids.

1995 ◽  
Vol 15 (8) ◽  
pp. 4497-4506 ◽  
Author(s):  
S A Wek ◽  
S Zhu ◽  
R C Wek
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Trna Synthetase ◽  
Amino Acid Starvation ◽  
Related Sequence ◽  
General Amino Acid Control ◽  
Trna Synthetases ◽  
Acid Control ◽  
Direct Regulator

Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.

scholarly journals Global Role of the Protein Kinase Gcn2 in the Human Pathogen Candida albicans

2005 ◽  
Vol 4 (10) ◽  
pp. 1687-1696 ◽  
Author(s):  
Hélène Tournu ◽  
Gyanendra Tripathi ◽  
Gwyneth Bertram ◽  
Susan Macaskill ◽  
Abigail Mavor ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Amino Acid Starvation ◽  
Transcriptional Level ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Eif2α Kinase ◽  
Starvation Conditions ◽  
Human Pathogen Candida Albicans

ABSTRACT The pathogen Candida albicans responds to amino acid starvation by activating pseudohyphal development and the expression of amino acid biosynthetic genes (GCN response). In Saccharomyces cerevisiae, the GCN response is dependent on Gcn2, which regulates the translation of the transcription factor Gcn4. Therefore, we examined the role of Gcn2 in C. albicans by using molecular, cellular, and genomic approaches. We show that C. albicans GCN2 encodes an eIF2α kinase, like its S. cerevisiae homologue. However, GCN4 appears to be regulated mainly at the transcriptional level in C. albicans. Furthermore, the inactivation of C. albicans Gcn2 only partially attenuates growth under amino acid starvation conditions and resistance to the histidine analogue 3-aminotriazole. Our comparison of the Gcn4 and Gcn2 regulons by transcript profiling reinforces the view that Gcn2 contributes to, but is not essential for, the activation of general amino acid control in C. albicans.

scholarly journals Insulin Signaling and the General Amino Acid Control Response

2008 ◽  
Vol 283 (28) ◽  
pp. 19229-19234 ◽  
Author(s):  
Sharon E. Malmberg ◽  
Christopher M. Adams
Keyword(s):  
Amino Acid ◽  
Insulin Signaling ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Control Response

scholarly journals The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253494
Author(s):  
Vera Cherkasova ◽  
James R. Iben ◽  
Kevin J. Pridham ◽  
Alan C. Kessler ◽  
Richard J. Maraia
Keyword(s):  
Amino Acid ◽  
Negative Regulator ◽  
Environmental Stress Response ◽  
Trna Processing ◽  
General Amino Acid Control ◽  
Leucine Uptake ◽  
Acid Control ◽  
La Protein ◽  
Control Response ◽  
Nuclear Exosome

The sla1+ gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4+ inhibits leucine uptake and growth of sla1Δ cells. Here, transcriptome analysis reveals that genes upregulated in sla1Δ cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4+ media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4+-effects, we isolated sla1Δ spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4+-sensitive leucine uptake linked to TOR. We show that 3H-leucine uptake by SSR-any1-F32V cells in NH4+-media is more robust than by sla1Δ cells. Moreover, F32V may alter any1+ function in sla1Δ vs. sla1+ cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.

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