Ribosome Binding Site Region Research Articles

Characterization and implications of prokaryotic ribosome-binding sites across species

The ribosome-binding site (RBS) in the 5′ untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system. RBSs have been widely applied in regulating gene expression in various scenarios, including Gram-negative or Gram-positive bacteria. Here, we first rationally designed and constructed an RBS mutant library containing 66 RBSs. The strength of these RBSs in E. coli and C. glutamicum was characterized individually. The RBS strength spanned about 200 and 15 times in the two species, respectively. The strength of RBSs in C. glutamicum was generally lower than that of in E. coli. A total of 18 RBSs showed similar strength (within twofold differences) between the species in our study, and the correlation analysis of the strength of RBSs between E. coli and C. glutamicum (R2 = 0.7483) revealed that these RBSs can be used across species. The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species. The RBS characterized here can be used to precisely regulate gene expression in both hosts, and the characteristics of cross-species RBSs provide basic information for RBS rational design.

Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment.

It has been firmly established that organic osmolytes (compatible solutes) of halophilic Bacteria and Archaea have positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strain Halomonas elongata accumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins. For the construction of broad hoast range vector systems with fluorescent reporter proteins, we chose the salt-inducible promoter region of the ectoine gene cluster (promA). A closer inspection of the genetic background revealed that its combination of sigma 38 (σ38) and sigma 70 (σ70) promoters was followed by a weak ribosomal binding site (RBS). This inspired a systematic approach for the construction of a promA-based vector series with a synthetic RBS region using the RBS Calculator v2.0, which resulted in a greatly improved salt-dependent expression—even in a deletion construct lacking the σ38 promoter. To expand the application range of this expression system, we looked further into the possible export of recombinant proteins into the periplasm. Both sec and tat leader sequences from H. elongata proved to be suitable for directed periplasmic transport into an extreme environment of freely selectable ionic strength.

Sintezna biologija za proizvodnjo biobutanola

In 2015, the first-ever Slovenian high school team competed at the iGEM (international Genetically Engineered Machine) competition in synthetic biology. The team was carefully selected from a list of candidates proposed by their high school teachers of chemistry and biology. Composed of eight students from 7 high schools from across the country, the team split into two groups, working in two institutions but with regular common meetings. The group of four students who worked at the National Institute of Chemistry focused on biotechnological production and the second group was preparing genetic constructs at the University of Ljubljana Faculty of Chemistry and Chemical Technology. The central problem that students tangled was the conversion of C-4 acids that are side-products of anaerobic waste degradation, into the corresponding alcohol. In the biotechnological part of the project students tested butanoic acid production in an anaerobic fermentation broth using a 15-channel computer-controlled bioreactor system, configured to allow online analysis of gaseous products (by GC) and frequent analysis of dissolved compounds (by HPLC). Next, at optimal butanoic acid production conditions (c = 1 g/L butanoic acid) growth of E. coli was examined in terms of medium composition, temperature and pH in order to obtain optimal operational parameters for biotechnological transformation of butanoic acid to butanol. Overall, E. coli growth was analysed in 72 different growth media in microtiter plates. Students followed inhibition of E. coli growth by butanol, acetone and isobutanol in the presence of butanoic acid and glycerol. The synthetic biology group started with PCR amplification of three genes ( ctf A and ctf B encoding two polypeptide chains of Co-A transferase, and bdh B encoding butyraldehide dehydrogenase for a two.stage conversion of butyril CoA to butanol) from anaerobic microbial community. Amplification failed, probably due to contaminants in the broth and a low number of bacteria harbouring these genes. Consequently, synthetic genes were designed with appended sequences for easier cloning, for a hexahistidine tag for detection of recombinant proteins using specific antibodies. Synthetic genes were inserted into pSB1C3 vectors and for each of them a strong promoter- ribosome binding site region was inserted in front of the enzyme coding region. Such combined constructs were further transferred into vectors with double terminators of transcription. In total, 9 different DNA constructs were prepared and deposited in the Registry of biological parts. All final expression constructs efficiently directed production of recombinant enzymes in E. coli DH5α under aerobic conditions. The giant jamboree of competing teams took place in Boston, USA, from September 24 to 28 with more than 260 teams from around the globe. In the high school track the Slovenian team presented the project entitled “From waste to fuel: Reprogrammed E. coli for sustainable production of biobutanol from butanoic acid”. The project received high recognition, as it was awarded a gold medal for completed tasks and was nominated among five best teams in categories Best Integrated Human Practices, Best Wiki, Best Presentation and Best High School Project.

High activity expression of d-amino acid oxidase in Escherichia coli by the protein expression rate optimization

Two categories of expression systems with different promoters and substitutive ribosome binding site region (RBS) were constructed in order to improve the soluble expression of d-amino acid oxidase (DAAO) basing on the hypothesis that the optimal promoter and suitable RBS would provide the recombinant expression system with better matched expression rate, which served as key factor to the heterogenous synthesis of soluble protein. The results showed that with rational promoter recombination and delicate RBS substitution, significant increase of DAAO activity (20-fold) was obtained for strain JM105/pGEMKT-Tac-R-DAAO over the previously constructed strain BL21(DE3)/pET-DAAO. Furthermore, similar optimization strategy proved feasible in the active expression of other enzymes such as glutaryl-7-aminocephalosporanic acid acylase (GCA) and N-Carbamyl-d-amino acid amidohydrolase (d-Case).

Effects of codon sequence on the dynamics of genetic networks

In prokaryotes, the rate at which codons are translated varies from one codon to the next. Using a stochastic model of transcription and translation at the nucleotide and codon levels, we investigate the effects of the codon sequence on the dynamics of protein numbers. For sequences generated according to the codon frequencies in Escherichia coli, we find that mean protein numbers at near equilibrium differ with the codon sequence, due to the mean codon translation efficiencies, in particular of the codons at the ribosome binding site region. We find close agreement between these predictions and measurements of protein expression levels as a function of the codon sequence. Next, we investigate the effects of short codon sequences at the start/end of the RNA sequence with linearly increasing/decreasing translation efficiencies, known as slow ramps. The ramps affect the mean, but not the fluctuations, in proteins numbers by affecting the rate of translation initiation. Finally, we show that slow ramps affect the dynamics of small genetic circuits, namely, switches and clocks. In switches, ramps affect the frequency of switching and bias the robustness of the noisy attractors. In repressilators, ramps alter the robustness of periodicity. We conclude that codon sequences affect the dynamics of gene expression and genetic circuits and, thus, are likely to be under selection regarding both mean codon frequency as well as spatial arrangement along the sequence.

Identification of a Novel Keyhole Phenotype in Double-Disk Diffusion Assays of Clindamycin-Resistant Erythromycin-Sensitive Strains of Streptococcus agalactiae

Our objective was to characterize 46 unique, erythromycin-sensitive, and clindamycin-resistant Streptococcus agalactiae strains from S. Korea that displayed a novel phenotype in double-disk diffusion assay. We used polymerase chain reaction to determine presence of erythromycin and clindamycin resistance genes, disc diffusion assays to determine resistance phenotype, and microbroth dilution to determine minimal inhibitory concentration. We detected a novel phenotype in the double-disk diffusion assay for inducible resistance among 46 S. agalactiae strains that were both erythromycin sensitive and clindamycin resistant. Thirty-two strains with the novel phenotype tested positive for erm(B) by DNA-DNA hybridization; sequencing of the erm(B) gene revealed mutations in the ribosomal binding site region in the erm(B) open reading frame, which is consistent with a lack of erythromycin resistance phenotype. Although identified from patients at multiple hospitals, genotyping suggested that the strains are closely related. The new phenotype shows increased sensitivity to clindamycin in the presence of erythromycin.

SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA

SsrA RNA (small stable RNA A), also known as tmRNA and 10Sa RNA, functions both as tRNA and mRNA through its unique structure. The carotenoid pigment is the eponymous feature of human pathogen Staphylococcus aureus. Here, we found that the pigment of the mutant strain with ssrA deletion was increased. Furthermore, it was demonstrated that ssrA could act as an antisense RNA aside from its well-known biological function, and crtMN, encoding two essential enzymes for the pigment synthesis, was identified as target of ssrA. Further investigation showed ssrA could specifically base pair with the RBS (ribosomal binding site) region of the crtMN mRNA. Our results revealed a new mechanism by which ssrA regulated the biosynthesis of pigment in S. aureus.

High Soluble Expression of d-Amino Acid Oxidase in Escherichia coli Regulated by a Native Promoter

To express high-active soluble D-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (P(Hase)), was constructed. A D-amino acid oxidase gene (dao) was ligated with the P(Hase) and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl beta-D-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m(3) fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 degrees C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 degrees C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.

Transcriptional and Translational Control of the Salmonella fliC Gene

The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5' untranslated region (5'UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5'UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5' end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC-dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the -10 and -35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5' end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5'UTR. Motility-specific mutants resulted from base substitutions only in the fliC-coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum.

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.

Use of a short A/T-rich cassette for enhanced expression of cloned genes in Escherichia coli.

A short (43-bp) A/T-rich stretch of DNA located in the intergenic region between the baiA2 and baiF genes from Eubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressed Eubacterium sp. strain VPI 12708 genes cloned in Escherichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from a baiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing the baiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with the baiF and baiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.

E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism.

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed.

Overproduction and purification of the ω subunit of Escherichia coli RNA polymerase

This paper reports the construction of plasmids which direct the overproduction of the w subunit of Escherichia coli RNA polymerase and the subsequent purification of ω. Useful overproduction is achieved only if the natural ribosomal binding site region of rpoZ is replaced with the ribosomal binding site region of bacteriophage T7 gene 10. Overproduction is directed by T7 RNA polymerase which is provided on a separate plasmid. ω is purified by three column steps either from the insoluble inclusion body fraction or from the soluble fractions of lysates. The final yield is approximately 2 mg ω per 10 g cells wet wt. Additionally, we found that recombinant ω is readily cleaved by an endogenous protease. Sequence analysis of the most prevalent proteolytic fragment suggested that the protease responsible was the product of the ompT gene. Cleavage of ω is greatly reduced in ompT − strains.

Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein

The translational efficiency of the coat protein gene of phage MS2 has been examined in vivo with respect to neighbouring sequences. The cloned MS2 DNA has been gradually shortened starting at the 5′ or 3′ terminus, and its effect on coat protein synthesis monitored. Removal of the 3'-terminal sequences had little influence. In contrast, the gradual removal of the 5′-terminal region profoundly reduces translation. Long before the ribosomal binding site (RBS) of the coat protein (CP) gene is reached, the yield of CP has dropped by one order of magnitude. Functional half-lives of the various messengers were found not to be significantly different. Available evidence indicates that the secondary structure of the RBS in native and shortened MS2 RNA is identical. We infer that important determinants for ribosome recognition lie 5′ to the RBS region of the MS2 RNA coat gene.

Increased expression of a cloned gene by local mutagenesis of its promoter and ribosome binding site.

A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E. coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased beta-galactosidase synthesis, were selected for DNA sequencing. One clone has a G leads to A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C leads to T and T leads to C) between the Shine-Dalgarno sequence and initiation codon which raise expression of beta-galactosidase two-fold. A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present. These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene.

The base sequence of the ribosome binding site region of the Gram-positive Staphylococcus aureus beta-lactamase gene has been determined. The leader peptide sequence of 24 amino acids which precedes the NH2 terminus of extracellular S. aureus beta-lactamase has also been established. This initiation site possesses two unique features not observed for most initiation sites recognized by Escherichia coli ribosomes. A novel initiation codon, UUG, initiates protein synthesis with methionine; and a very strong Shine-Dalgarno complementarity containing five G-C base pairs precedes the UUG initiation codon. The strong Shine-Dalgarno complementarity may explain the reduced translational dependence on initiation factor IF-3 function that has been observed for the beta-lactamase mRNA and other mRNAs from Gram-positive bacteria. We suggest that this extent of complementarity between the mRNA and this extent of complementarity between the mRNA and the ribosome may be a requirement for efficient initiation by Bacillus subtilis and other Gram-positive ribosomes, and may provide the basis for the observed inability of the Gram-positive systems to translate most of the mRNAs from Gram-negative bacteria.