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Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

ABSTRACT: A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC).Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (?G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (?G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%.We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

SUBMITTER: Lamping E

PROVIDER: S-EPMC3765126 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

Lamping Erwin E Niimi Masakazu M Cannon Richard D RD

Microbial cell factories 20130729

<h4>Background</h4>A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirectin ...[more]

PMID: 23895661

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Project description:BackgroundA virus was isolated from diseased turbot Scophthalmus maximus in China. Biophysical and biochemical assays, electron microscopy, and genome electrophoresis revealed that the virus belonged to the genus Aquareovirus, and was named Scophthalmus maximus reovirus (SMReV). To the best of our knowledge, no complete sequence of an aquareovirus from marine fish has been determined. Therefore, the complete characterization and analysis of the genome of this novel aquareovirus will facilitate further understanding of the taxonomic distribution of aquareovirus species and the molecular mechanism of its pathogenesis.ResultsThe full-length genome sequences of SMReV were determined. It comprises eleven dsRNA segments covering 24,042 base pairs and has the largest S4 genome segment in the sequenced aquareoviruses. Sequence analysis showed that all of the segments contained six conserved nucleotides at the 5' end and five conserved nucleotides at the 3' end (5'-GUUUUA ---- UCAUC-3'). The encoded amino acid sequences share the highest sequence identities with the respective proteins of aquareoviruses in species group Aquareovirus A. Phylogenetic analysis based on the major outer capsid protein VP7 and RNA-dependent RNA polymerase were performed. Members in Aquareovirus were clustered in two groups, one from fresh water fish and the other from marine fish. Furthermore, a fusion associated small transmembrane (FAST) protein NS22, which is translated from a non-AUG start site, was identified in the S7 segment.ConclusionsThis study has provided the complete genome sequence of a novel isolated aquareovirus from marine fish. Amino acids comparison and phylogenetic analysis suggested that SMReV was a new aquareovirus in the species group Aquareovirus A. Phylogenetic analysis among aquareoviruses revealed that VP7 could be used as a reference to divide the aquareovirus from hosts in fresh water or marine. In addition, a FAST protein with a non-AUG start site was identified, which partially contributed to the cytopathic effect caused by the virus infection. These results provide new insights into the virus-host and virus-environment interactions.

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Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

Publications

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

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