Amino acid starvation and colicin D treatment induce A-site mRNA cleavage in Escherichia coli

Escherichia coli possesses a unique RNase activity that cleaves stop codons in the ribosomal aminoacyl-tRNA binding site (A-site) during inefficient translation termination. This A-site mRNA cleavage allows recycling of arrested ribosomes by facilitating recruitment of the tmRNA•SmpB ribosome rescue system. To test whether A-site nuclease activity also cleaves sense codons, we induced ribosome pausing at each of the six arginine codons using three strategies; rare codon usage, arginine starvation, and inactivation of arginine tRNAs with colicin D. In each instance, ribosome pausing induced mRNA cleavage within the target arginine codons, and resulted in tmRNA-mediated SsrA-peptide tagging of the nascent polypeptide. A-site mRNA cleavage did not require the stringent factor ppGpp, or bacterial toxins such as RelE, which mediates a similar nuclease activity. However, the efficiency of A-site cleavage was modulated by the identity of the two codons immediately upstream (5′ side) of the A-site codon. Starvation for histidine and tryptophan also induced A-site cleavage at histidine and tryptophan codons, respectively. Thus, A-site mRNA cleavage is a general response to ribosome pausing, capable of cleaving a variety of sense and stop codons. The induction of A-site cleavage during amino acid starvation suggests this nuclease activity may help to regulate protein synthesis during nutritional stress.     

An improved strategy for efficient expression and purification of soluble HIV-1 tat protein in <Emphasis Type="Italic">E.coli</Emphasis>

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.     

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

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.     

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)

The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).     

tRNA residues evolved to promote translational accuracy

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA^Ala_GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA^Ala_GGC sequences. This suggests that the sequences of bacterial tRNA^Ala_GGC have evolved to avoid reading incorrect codons.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Simple and Efficient Method for Heterologous Expression of Clostridial Proteins

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.     

Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA^Pyl pairs and cross recognition between nonsense codons and various tRNA^Pyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N^ε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.     

Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli

Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_gnaD and Tac_gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_gnaD and Tac_gnaD gene sequences revealed that the 5'-end of the Sso_gnaD sequence was rich in AGA(arg) and ATA(Ile) codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.     

A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 × 10⁻⁵ to a high of 6.9 × 10⁻⁴ per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.     

Systematic Comparison of Strategies to Achieve Soluble Expression of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Recombinant Proteins in <Emphasis Type="Italic">E. coli</Emphasis>

Constructs containing partial coding sequences of myosin A, myosin B, and glideosome-associated protein (50 kDa) of Plasmodium falciparum were used to challenge several strategies designed in order to improve the production and solubility of recombinant proteins in Escherichia coli. Assays were carried out inducing expression in a late log phase culture, optimizing the inductor concentration, reducing the growth temperature for induced cultures, and supplementing additives in the lysis buffer. In addition, recombinant proteins were expressed as fusion proteins with three different tags (6His, GST, and MBP) in four different E. coli strains. We found that the only condition that consistently produced soluble proteins was the use of MBP as a fusion tag, which became a valuable tool for detecting the proteins used in this study and did not caused any interference in protein–protein interaction assays (Far Western Blot). Besides, we found that BL21-pG-KJE8 strain did not improve the solubility of any of the recombinant protein produced, while the BL21-CodonPlus(DE3)-RIL strain improved the expression of some of them independent of the rare codon content. Proteins with rare codons occurring at high frequencies (»?10%) were expressed efficiently in strains that do not supplement tRNAs for these triplets.     

Modulation of lambda integrase synthesis by rare arginine tRNA

Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.     

Molecular evolution of bacteriophages: Discrete patterns of codon usage in T4 genes are related to the time of gene expression

Summary Patterns of codon usage in certain coliphages are adapted to expression inEscherichia coli. Bacteriophage T4 may be an exception to test the rule, as it produces eight tRNAs with specificities that are otherwise rare inE. coli. A database of all known T4 DNA sequences has been compiled, comprising 174 genes and a total of 115 kb (approximately 70% of the T4 genome). Codon usage has been examined in all T4 genes; some of these are known to be expressed before, and some after, the production of phage tRNAs. The results show two different patterns of codon usage: by comparison with the early genes, the late genes exhibit a shift in preference toward those codons recognized by the phage-encoded tRNAs. The T4 tRNAs translate A-ending codons, and it is possible that the phage acquired the tRNA genes because the mutation bias of the T4 DNA polymerase forces the T4 genome toward A+T-richness.Presented at the NATO Advanced Workshop on Genome Organization and Evolution, held in Spetses, Greece, September 1990     

Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs

There exists a similarity among the synonymous codon choice patterns of the yeast nuclear genes that have been sequenced thus far although these genes encode different types of protein molecules, and the patterns are significantly different from those of Escherichia coli genes. Based on constraints caused by the availability of E. coli transfer RNAs and the nature of their codon recognition related to the modified nucleotides at the anticodon wobble position, the characteristic patterns of synonymous codon choice commonly found for E. coli genes have been almost completely explained (Ikemura, 1981a, b). In the present paper, tRNAs of the yeast Saccharomyces cerevisiae were separated by two-dimensional polyacrylamide gel electrophoresis and the relative abundance of purified tRNA molecules was measured on the basis of molecular numbers in cells. A strong correlation between tRNA abundance and codon choice was found for each nuclear gene of yeast, but the correlation was less significant for 2μ plasmid genes. According to the criteria proposed for E. coli genes (Ikemura, 1981b) the order of codon preference in yeast nuclear genes was predicted based on the abundance of yeast isoaccepting tRNAs and on the nature of the modified nucleotides at their anticodons. Clear correlations between predictions and the actual preferences among synonymous codons were revealed, indicating that the codon choices in yeast genes are also constrained by a combination of tRNA availability and nature of its codon recognition. Then the difference in synonymous codon use between the two organisms can be attributed to the difference in these two factors.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.     

Efficient expression of gene variants that harbour AGA codons next to the initiation codon

In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons. The mixed combinations of AGA and each of the CGN codons usually resulted in efficient rates of lacZ expression independently of the peptidyl-tRNA propensity to dissociate from the ribosome. Thus, the variant harboring the pair of AGA codons was expressed as efficiently as the variant carrying a pair of AAA codons in the same positions, a configuration reported as one of the most common and efficient for gene expression. We explain these results assuming that the presence of adenines in these early positions enhance gene expression. As expected, specific mRNA levels correlated with the intensity of lacZ expression for each variant. However, the induction of lacZ AGA AGA gene in pth cells accumulated peptidyl-tRNA^Arg4 as well as a short 5′-proximal lacZ mRNA fragment suggesting ribosome stalling due to depletion of aminoacylated-tRNA^Arg4.     

New recombinant producer of human ω-amidase based on <Emphasis Type="Italic">Escherichia coli</Emphasis>

A new artificial gene encoding human ω-amidase (Nit2) adapted for highly efficient expression in E. coli has been established. A pQE-Nit2 plasmid construct controlled by the T5 promoter has been engineered for its expression. The nit2 gene within the pQE-Nit2 construct has optimized codon usage and an artificial 6His-tag sequence inserted directly after the ATG initiation codon. This tag provides the possibility of single-step purification of a product via metal chelate chromatography. The codon-usage optimization involves the inclusion of several codons of extremely rare occurrence in natural E. coli ORFs within a 30 a.a-long N-terminal region. Other codons included in the N-terminus have moderate occurrence in E. coli. The subsequent sequence of the artificial gene has been composed of the most frequently occurring codons in E. coli. The recombinant producer based on the pQE-Nit2 construct allowed purification of the enzyme with an activity of 6.2 ± 0.2 μmol/min/mg protein, which corresponds to or slightly exceeds the specific activity of rat liver Nit2. The omega-amidase preparation is necessary for the screening of potential inhibitors that can be used as candidate drugs to cure hyperammonemia disorders in liver pathologies and oncological diseases.