scholarly journals Translational Features of Human Alpha 2b Interferon Production in Escherichia coli

2004 ◽  
Vol 70 (8) ◽  
pp. 5033-5036 ◽  
Author(s):  
C. A. Valente ◽  
D. M. F. Prazeres ◽  
J. M. S. Cabral ◽  
G. A. Monteiro
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Codon Usage ◽  
Gene Variants ◽  
Interferon Production ◽  
Mrna Secondary Structure ◽  
Translation Machinery ◽  
E Coli

ABSTRACT The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5′ end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.

scholarly journals A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Adi Oron-Gottesman ◽  
Martina Sauert ◽  
Isabella Moll ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Genetic Code ◽  
Open Reading Frames ◽  
Translation Machinery ◽  
E Coli ◽  
Universal Characteristic ◽  
Living Organisms ◽  
Reading Frames

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. IMPORTANCE The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli , under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs.

scholarly journals Stress-Induced MazF-Mediated Proteins in Escherichia coli

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Akanksha Nigam ◽  
Tamar Ziv ◽  
Adi Oron-Gottesman ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Proteomic Analysis ◽  
Normal Growth ◽  
Growth Conditions ◽  
Translation Machinery ◽  
Content Type ◽  
E Coli ◽  
Adverse Conditions ◽  
Almost All ◽  
Induced Proteins

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions. IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

scholarly journals Codon optimization of Col H gene encoding Clostridium histolyticum collagenase to express in Escherichia coli

2014 ◽  
Author(s):  
Hamzeh Alipour ◽  
Abbasali Raz ◽  
Navid Dinparast Djadid ◽  
Abbas Rami ◽  
Seyed Mohammad Amin Mahdian
Keyword(s):  
Escherichia Coli ◽  
Codon Usage ◽  
Codon Optimization ◽  
Coding Region ◽  
Gene Encoding ◽  
E Coli ◽  
Clostridium Histolyticum ◽  
Optimal Codons ◽  
H Gene ◽  
Clostridium Histolyticum Collagenase

A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, prove not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. As regards the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). The coding region of mature Col H gene was optimized according to the codon usage of E. coli using Gene Designer software (DNA 2.0). The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. Similarly, the higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. As has been reported elsewhere, the findings from this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. In spite of that, experimental research is needed to confirm the improvement.

Modification of mRNA secondary structure and alteration of the expression of human interferon α1 in Escherichia coli

Gene ◽  
1987 ◽  
Vol 58 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Nancy Lee ◽  
Zhang Sun-Qu ◽  
Joseph Cozzitorto ◽  
Yang Jin-Shui ◽  
Douglas Testa
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Human Interferon ◽  
Mrna Secondary Structure

A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in E. coli

2017 ◽  
Vol 114 (8) ◽  
pp. 1753-1761 ◽  
Author(s):  
Varnika Roy ◽  
Robert Roth ◽  
Mark Berge ◽  
Rajesh Chitta ◽  
Sucheta Vajrala ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Mrna Secondary Structure ◽  
E Coli ◽  
Bicistronic Vector

scholarly journals Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

2016 ◽  
Author(s):  
Michael G. Napolitano ◽  
Matthieu Landon ◽  
Christopher J. Gregg ◽  
Marc J. Lajoie ◽  
Lakshmi N. Govindarajan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Secondary Structure ◽  
Binding Site ◽  
Amino Acid Identity ◽  
Mrna Secondary Structure ◽  
Acid Identity ◽  
Genetic Codes ◽  
Genome Wide ◽  
Ribosomal Binding Site

AbstractThe degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as non-coding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU, but the remaining thirteen “recalcitrant” AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multi-dimensional “safe replacement zone” (SRZ) within which alternative codons are more likely to be viable. To further evaluate synonymous and non-synonymous alternatives to essential AGRs, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild type allele, allowing us to exhaustively evaluate the fitness impact of all 64 codon alternatives. Using this method, we confirmed relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.Significance StatementThis work presents the genome-wide replacement of all rare AGR arginine codons in the essential genes of Escherichia coli with synonymous CGN alternatives. Synonymous codon substitutions can lethally impact non-coding function by disrupting mRNA secondary structure and ribosomal binding site-like motifs. Here we quantitatively define the range of tolerable deviation in these metrics and use this relationship to provide critical insight into codon choice in recoded genomes. This work demonstrates that genome-wide removal of AGR is likely to be possible, and provides a framework for designing genomes with radically altered genetic codes.

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