Shine-Dalgarno Research Articles

The Effect of Translation Promoting Site (TPS) on Protein Expression in E. coli Cells.

The study of translation initiation in prokaryotes assumes that there should be a mechanism different from the canonical model, which postulates the formation of the pre-initiation complex through the interaction of the Shine-Dalgarno sequence (SD) at the 5'-end of mRNA and the anti-Shine-Dalgarno site at the 3'-end of 16S rRNA. In this paper we've studied the effect of TPS (Translation-initiation Promoting Site) on β-glucuronidase expression in E. coli cells at different cultivation temperatures. The examined leader sequences were cloned into the pET23c plasmid upstream the β-glucuronidase gene; protein expression was performed in E. coli BL21 (DE3) cells. β-glucuronidase activity was measured in bacterial cell extracts via paranitrophenyl b-D-glucuronide assay. The quantity of expressed protein was measured by Western blotting with following densitometry. It was shown that TPS increases the level of protein expression at stressful conditions (10°C and 44°C) 5-8 times compared to control. The combination of TPS and SD sites in the 5'-leader sequence of the mRNA created an enhancer that increased the expression level 2-3.6 times compared to a single SD-sequence. Based on the obtained data and the computer modeling of interaction between 16S rRNA and TPS, we proposed an alternative variation of prokaryotic translation initiation.

Influence of the spacer region between the Shine-Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli.

SummaryTranslation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next‐generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100‐fold range.

Transcriptional and Translational Leakiness in Expression of a Fungal Cyclopropane Fatty Acid Synthase in the Escherichia coli pET-vector Expression System

We expressed the putative cyclopropane synthase Cfs1 from the mushroom Coprinopsis cinerea and the native bacterial Cfa enzyme in Escherichia coli as fusion proteins with an Nterminal 10xHis-tag and a proteolytic Factor Xa site, using the commercial pET-16b vector with the T7lac promoter for expression. Proteomics detected both proteins with high Mascot scores via trypsin-digested peptides in total cytoplasmic protein samples from transformed BL21(DE3) bacteria, induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) for T7 RNA polymerase production from theT7 phage gene 1 present in the host genome under control by the lacUV5 promoter. In lower amounts, Cfs1 and Cfa were also found in protein samples from non-induced cells based on the known transcriptional leakiness of the pET-system in expression of T7 RNA polymerase. As a novelty, the results on Cfs1 from proteomics suggest that the sequence at the pET-16b-cfs1 fusion point CTCGAGGAT CCTATG (BamHI cloning site in italic, native cfs1 ATG start codon in bold) serves in transcripts as a minor RBS (ribosome binding site) to inadvertently initiate protein synthesis on the native cfs1 start codon. The first peptide MPAHHHPSSSAPCVSFPSSSK of the native Cfs1 protein was found in E. coli which is only possible if the normal cfs1 ATG was recognized as the start codon in protein biosynthesis. The GAGG-stretch in the above DNA sequence with a 7-bases-aligned spacing to the native cfs1 start codon features a partial Shine-Dalgarno (SD) sequence.

Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing.

Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in E. coli show no correlation between the strength of an mRNA's SD motif and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for ribosomes to determine where initiation occurs, though they do affect how efficiently initiation occurs.

Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression. They are model systems for analyzing RNA-ligand interactions and are established targets for antibacterial agents. Many studies have analyzed the ligand-binding properties of riboswitches, but this work has outpaced our understanding of the underlying chemical pathways that govern riboswitch-controlled gene expression. To address this knowledge gap, we prepared 15 mutants of the preQ1-II riboswitch-a structurally and biochemically well-characterized HLout pseudoknot that recognizes the metabolite prequeuosine1 (preQ1). The mutants span the preQ1-binding pocket through the adjoining Shine-Dalgarno sequence (SDS) and include A-minor motifs, pseudoknot-insertion helix P4, U·A-U base triples, and canonical G-C pairs in the anti-SDS. As predicted-and confirmed by in vitro isothermal titration calorimetry measurements-specific mutations ablated preQ1 binding, but most aberrant binding effects were corrected by compensatory mutations. In contrast, functional analysis in live bacteria using a riboswitch-controlled GFPuv-reporter assay revealed that each mutant had a deleterious effect on gene regulation, even when compensatory changes were included. Our results indicate that effector binding can be uncoupled from gene regulation. We attribute loss of function to defects in a chemical interaction network that links effector binding to distal regions of the fold that support the gene-off RNA conformation. Our findings differentiate effector binding from biological function, which has ramifications for riboswitch characterization. Our results are considered in the context of synthetic ligands and drugs that bind tightly to riboswitches without eliciting a biological response.

Protein unties the pseudoknot: S1-mediated unfolding of RNA higher order structure.

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5′ UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.

Unique Shine-Dalgarno Sequences in Cyanobacteria and Chloroplasts Reveal Evolutionary Differences in Their Translation Initiation.

Microorganisms require efficient translation to grow and replicate rapidly, and translation is often rate-limited by initiation. A prominent feature that facilitates translation initiation in bacteria is the Shine–Dalgarno (SD) sequence. However, there is much debate over its conservation in Cyanobacteria and in chloroplasts which presumably originated from endosymbiosis of ancient Cyanobacteria. Elucidating the utilization of SD sequences in Cyanobacteria and in chloroplasts is therefore important to understand whether 1) SD role in Cyanobacterial translation has been reduced prior to chloroplast endosymbiosis or 2) translation in Cyanobacteria and in plastid has been subjected to different evolutionary pressures. To test these alternatives, we employed genomic, proteomic, and transcriptomic data to trace differences in SD usage among Synechocystis species, Microcystis aeruginosa, cyanophages, Nicotiana tabacum chloroplast, and Arabidopsis thaliana chloroplast. We corrected their mis-annotated 16S rRNA 3′ terminus using an RNA-Seq-based approach to determine their SD/anti-SD locational constraints using an improved measurement DtoStart. We found that cyanophages well-mimic Cyanobacteria in SD usage because both have been under the same selection pressure for SD-mediated initiation. Whereas chloroplasts lost this similarity because the need for SD-facilitated initiation has been reduced in plastids having much reduced genome size and different ribosomal proteins as a result of host-symbiont coevolution. Consequently, SD sequence significantly increases protein expression in Cyanobacteria but not in chloroplasts, and only Cyanobacterial genes compensate for a lack of SD sequence by having weaker secondary structures at the 5′ UTR. Our results suggest different evolutionary pressures operate on translation initiation in Cyanobacteria and in chloroplast.

Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria.

In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine–Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position –3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved β-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.

Small RNA NcS27 co-regulates utilization of carbon sources in Burkholderia cenocepacia J2315.

Small non-coding sRNAs have versatile roles in regulating bacterial metabolism. Four short homologous Burkholderia cenocepacia sRNAs strongly expressed under conditions of growth arrest were recently identified. Here we report the detailed investigation of one of these, NcS27. sRNA NcS27 contains a short putative target recognition sequence, which is conserved throughout the order Burkholderiales. This sequence is the reverse complement of the Shine-Dalgarno sequence of a large number of genes involved in transport and metabolism of amino acids and carbohydrates. Overexpression of NcS27 sRNA had a distinct impact on growth, attenuating growth on a variety of substrates such as phenylalanine, tyrosine, glycerol and galactose, while having no effect on growth on other substrates. Transcriptomics and proteomics of NcS27 overexpression and silencing mutants revealed numerous predicted targets changing expression, notably of genes involved in degradation of aromatic amino acids phenylalanine and tyrosine, and in transport of carbohydrates. The conserved target recognition sequence was essential for growth phenotypes and gene expression changes. Cumulatively, our data point to a role of NcS27 in regulating the shutdown of metabolism upon nutrient deprivation in B. cenocepacia. We propose Burkholderiadouble-hairpin sRNA regulator bdhR1 as designation for ncS27.

Characterization and mechanism of the effects of Mg\u2013Fe layered double hydroxide nanoparticles on a marine bacterium: new insights from genomic and transcriptional analyses

BackgroundLayered double hydroxides (LDHs) have received widespread attention for their potential applications in catalysis, polymer nanocomposites, pharmaceuticals, and sensors. Here, the mechanism underlying the physiological effects of Mg–Fe layered double hydroxide nanoparticles on the marine bacterial species Arthrobacter oxidans KQ11 was investigated.ResultsIncreased yields of marine dextranase (Aodex) were obtained by exposing A. oxidans KQ11 to Mg–Fe layered double hydroxide nanoparticles (Mg–Fe-LDH NPs). Furthermore, the potential effects of Mg–Fe-LDH NPs on bacterial growth and Aodex production were preliminarily investigated. A. oxidans KQ11 growth was not affected by exposure to the Mg–Fe-LDH NPs. In contrast, a U-shaped trend of Aodex production was observed after exposure to NPs at a concentration of 10 μg/L–100 mg/L, which was due to competition between Mg–Fe-LDH NP adsorption on Aodex and the promotion of Aodex expression by the NPs. The mechanism underling the effects of Mg–Fe-LDH NPs on A. oxidans KQ11 was investigated using a combination of physiological characterization, genomics, and transcriptomics. Exposure to 100 mg/L of Mg–Fe-LDH NPs led to NP adsorption onto Aodex, increased expression of Aodex, and generation of a new Shine-Dalgarno sequence (GGGAG) and sRNAs that both influenced the expression of Aodex. Moreover, the expressions of transcripts related to ferric iron metabolic functions were significantly influenced by treatment.ConclusionsThese results provide valuable information for further investigation of the A. oxidans KQ11 response to Mg–Fe-LDH NPs and will aid in achieving improved marine dextranase production, and even improve such activities in other marine microorganisms.

Conservation of transcriptional elements in the obligate symbiont of the whitefly Bemisia tabaci.

BackgroundBacterial symbiosis is widespread in arthropods, especially in insects. Some of the symbionts undergo a long-term co-evolution with the host, resulting in massive genome decay. One particular consequence of genome decay is thought to be the elimination of transcriptional elements within both the coding region and intergenic sequences. In the whitefly Bemisia tabaci species complex, the obligate symbiont Candidatus Portiera aleyrodidarum is of vital importance in nutrient provision, and yet little is known about the regulatory capacities of it.MethodsPortiera genomes of two whitefly species in China were sequenced and assembled. Gene content of these two Portiera genomes was predicted, and then subjected to Kyoto Encyclopedia of Genes and Genomes pathway analysis. Together with two other Portiera genomes from whitefly species available previously, four Portiera genomes were utilized to investigate regulatory capacities of Portiera, focusing on transcriptional elements, including genes related with transcription and functional elements within the intergenic spacers.ResultsComparative analyses of the four Portiera genomes of whitefly B. tabaci indicate that the obligate symbionts Portiera is similar in different species of whiteflies, in terms of general genome features and possible functions in the biosynthesis of essential amino acids. The screening of transcriptional factors suggests compromised ability of Portiera to regulate the essential amino acid biosynthesis pathways. Meanwhile, thermal tolerance ability of Portiera is indicated with the detection of a σ32 factor, as well as two predicted σ32 binding sites. Within intergenic spacers, functional elements are predicted, including 37 Shine-Dalgarno sequences and 34 putative small RNAs.

A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA.

Post-transcriptional regulation plays important roles to fine-tune gene expression in bacteria. In particular, regulation of type I toxin-antitoxin (TA) systems is achieved through sophisticated mechanisms involving toxin mRNA folding. Here, we set up a genetic approach to decipher the molecular underpinnings behind the regulation of a type I TA in Helicobacter pylori. We used the lethality induced by chromosomal inactivation of the antitoxin to select mutations that suppress toxicity. We found that single point mutations are sufficient to allow cell survival. Mutations located either in the 5' untranslated region or within the open reading frame of the toxin hamper its translation by stabilizing stem-loop structures that sequester the Shine-Dalgarno sequence. We propose that these short hairpins correspond to metastable structures that are transiently formed during transcription to avoid premature toxin expression. This work uncovers the co-transcriptional inhibition of translation as an additional layer of TA regulation in bacteria.

Emergence of a "Cyclosome" in a Primitive Network Capable of Building "Infinite" Proteins.

We argue for the existence of an RNA sequence, called the AL (for ALpha) sequence, which may have played a role at the origin of life; this role entailed the AL sequence helping generate the first peptide assemblies via a primitive network. These peptide assemblies included “infinite” proteins. The AL sequence was constructed on an economy principle as the smallest RNA ring having one representative of each codon’s synonymy class and capable of adopting a non-functional but nevertheless evolutionarily stable hairpin form that resisted denaturation due to environmental changes in pH, hydration, temperature, etc. Long subsequences from the AL ring resemble sequences from tRNAs and 5S rRNAs of numerous species like the proteobacterium, Rhodobacter sphaeroides. Pentameric subsequences from the AL are present more frequently than expected in current genomes, in particular, in genes encoding some of the proteins associated with ribosomes like tRNA synthetases. Such relics may help explain the existence of universal sequences like exon/intron frontier regions, Shine-Dalgarno sequence (present in bacterial and archaeal mRNAs), CRISPR and mitochondrial loop sequences.

Optimizing Phage Translation Initiation

Phage as an anti-bacterial agent must be efficient in killing bacteria, and consequently needs to replicate efficiently. Protein production is a limiting step in replication in almost all forms of life, including phages. Efficient protein production depends on the efficiency of translation initiation, elongation and termination, with translation initiation often being rate limiting. Initiation signals such as Shine-Dalgarno (SD) sequences and start codon are decoded by anti-SD sequences and initiation tRNA, respectively. While the decoding machinery cannot be readily modified, the signals can be engineered to increase the efficiency of their decoding. Here I review our understanding of the translation machinery to facilitate the engineering of optimal translation initiation signals for facilitating the design of phage protein-coding genes, including 1) accurate characterization of the 3’ end of 16S rRNA by using RNA-Seq data, 2) identification of the optimal SD/aSD interaction, and 3) reduction of secondary structure in sequences flanking the start codon.

NEW INSIGHT INTO THE MECHANISM OF ACTION OF ε-ELEMENT ENHANCER IN E. COLI

Epsilon element (ε-element) is a complex translational enhancer capable of promoting the prokaryotic translation efficiency in combination with the Shine-Dalgarno (SD) sequence. However, the detailed mechanism of action of ε-element is unknown, and there is no single hypothesis to explain the available evidence. We here propose a new hypothesis for the mechanism of action of ε-element from the 5′-untranslated region (UTR) of the 10 gene of phage T7 during translation initiation in Escherichia coli. The mechanism of action was considered to be driven by the complementary interaction of ε-element (and its variation εII) with the 3′-end of E. coli 16S rRNA, specifically with the 45th hairpin region. This hypothesis was then applied to currently available data of the influence of ε-element on protein expression in bacterial cells. Several 5′-UTR sequences were constructed containing variations of ε-element, and their properties were predicted according to this new hypothesis and confirmed by measuring the protein expression level. We further applied temperature stresses (cold and heat shock) to these variants to examine their influence on protein expression controlled by various enhancers. Overall, the εII_SD and ε_SD enhancers could significantly increase the protein expression level in E. coli cells under cold shock, representing a 6–9-times increase relative to that obtained with the classical SD sequence.

A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis

ABSTRACTCsrA is a widely conserved, abundant small RNA binding protein that has been found in E. coli and other Gram-negative bacteria where it is involved in the regulation of carbon metabolism, biofilm formation and virulence. CsrA binds to single-stranded GGA motifs around the SD sequence of target mRNAs where it inhibits or activates translation or influences RNA processing. Small RNAs like CsrB or CsrC containing 13–22 GGA motifs can sequester CsrA, thereby abrogating the effect of CsrA on its target mRNAs. In B. subtilis, CsrA has so far only been found to regulate one target, hag mRNA and to be sequestered by a protein (FliW) and not by an sRNA. Here, we employ a combination of in vitro and in vivo methods to investigate the effect of CsrA on the small regulatory RNA SR1 from B. subtilis, its primary target ahrC mRNA and its downstream targets, the rocABC and rocDEF operons. We demonstrate that CsrA can promote the base-pairing interactions between SR1 and ahrC mRNA, a function that has so far only been found for Hfq or ProQ.Abbreviations: aa, amino acid; bp, basepair; nt, nucleotide; PAA, polyacrylamide; SD, Shine Dalgarno.

TRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

Role of Ribosome Recycling Factor in Translational Coupling as a Ribosome Releasing Factor

The objective of this presentation is to study the in vivo actions of ribosome recycling factor (RRF) and compare them with those found in vitro. RRF is known to catalyze three separate reactions: release of tRNA and mRNA from the post‐termination complex (PoTC), and splitting of the ribosome of the PoTC. To study the role of RRF in translational coupling in vivo, we used E. coli harboring temperature sensitive (ts) RRF and assayed by following downstream reading of translationally coupled ORF. At the non‐permissive temperature, ribosomes remain on the termination codon of the junction sequence of coupled ORFs and translate downstream ORF without the Shine‐Dalgarno (SD) sequence. The readings were in all three frames due to thermal frameshift at the termination codon. Introduction of sequence complementary to the 3′‐terminal of 16S rRNA into the region surrounding the junction sequence increased downstream reading. When upstream ORF was short, translation of the downstream reading was abolished, suggesting that the ribosomes released by RRF are moving toward the SD sequence of the upstream ORF. The thermal frame shift at the stop codon was also stopped by the upstream SD sequence. Our data suggest that the ribosome‐bound mRNA may take a secondary structure around the junction sequence. For in vitro studies, we used mRNA that incorporated different radioactively labeled amino acids based on the frameshift at the junction sequence, UAAUG, of two translationally coupled ORFs. In the absence of RRF, the ribosome stayed on the mRNA and translated in frame with the termination codon UAA. In the presence of RRF, amino acid incorporation occurred in frame with the start codon AUG. This suggests that RRF releases the ribosome from UAA and the released ribosome binds to AUG and begins translation. With the use of tethered, unsplittable ribosomes (Ribo‐T) in the in vitro system, we showed that complete ribosomal splitting is not required for the action of RRF in translational coupling. Therefore, the main role of RRF in translational coupling appears to be the release of ribosomes from mRNA.Support or Funding InformationSupported and funded by Creative Biomedical Research Institute (CBRI) in Philadelphia, Pennsylvania.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secretory production of xylanase in Corynebacterium glutamicum using its endogenous elements

We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.

Translation enhancement by a Dictyostelium gene sequence in Escherichia coli

Methods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed "translation enhancement by a Dictyostelium gene sequence" ("TED") in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3' end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.