Identification and in vivo expression of a prokaryotic-like ribosome recognition sequence upstream of the coat protein gene of potato virus X.

Conserved prokaryotic sequence motifs, distinct from the classic Shine-Dalgarno sequence, yet possessing homology to 16S rRNA in E. coli have been identified in a number of plant viruses. In this report, a similar Shine-Dalgarno-like motif located immediately upstream to the CP gene of potato virus X (PVX) was demonstrated to enable expression of a reporter gene in E. coli to approximately one third the level of a similar construct containing the classical Shine-Dalgarno sequence. Both PVX-specific CP and RNA transcripts were detected in chloroplasts purified from transgenic potato plants containing the PVX CP gene and corresponding leader sequence. Protoplasts generated from these transgenic plants were used to demonstrate that expression of the PVX CP from chloroplasts is possible. The implications of these results on the PVX infection cycle are discussed.

Epsilon as an initiator of translation of CAT mRNA in Escherichia coli.

Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.

Enhancing activity of epsilon in Escherichia coli and Agrobacterium tumefaciens cells.

Epsilon (epsilon) sequence is a bacterial enhancer of translation found in the bacteriophage T7 gene 10. It is believed that its enhancing effect of epsilon is due to a base-pairing with the nucleotides 458-467 from the helical domain 17 of Escherichia coli 16S rRNA. To prove this we have taken advantage of the difference of this domain in Agrobacterium tumefaciens and E. coli. To evaluate the significance of nucleotide complementarity for the enhancing activity of epsilon, a series of nucleotide sequences matching either E. coli or A. tumefaciens domain 17 are cloned in a binary expression vector in front of the chloramphenicol acetyltransferase (CAT) gene. The CAT assay shows that: (i) the epsilon in combination with an SD consensus sequence increases the yield of CAT in both microorganisms over that obtained with the SD alone; (ii) the epsilon sequence complementary to the A. tumefaciens domain 17 leads to a 2.71-fold increase in the yield of CAT in homologous cells but not in E. coli cells; (iii) the yield of CAT correlates with the free energy of base-pairing with the helical domain 17 in both microorganisms.

Non-enzymatic RNA hydrolysis promoted by the combined catalytic activity of buffers and magnesium ions.

Although Mg2+ is an important cofactor for the specific degradation of RNA by ribozymes, it is not considered as a typical chemical nuclease. In this study we show that in combination with common buffers such as tris(hydroxymethyl)aminomethane and sodium borate, Mg2+ is a powerful catalyst for the degradation of RNA. pH and temperature are found to be the principal factors for the efficient degradation of RNA. Whereas in Tris-HCl/Mg2+ the efficient cleavage starts at pH values higher than 7.5 and temperatures higher than 37 degrees C, in sodium borate RNA degradation begins at pH 7.0 and at 37 degrees C. RNA hydrolysis promoted under the combined catalytic activity of buffer/Mg2+ results in partially degraded RNA and negligible amounts of acid-soluble material. Reaction is insensitive to the concentration of monovalent cations but is completely prevented by chelating agents (EDTA and citrate) at concentrations exceeding that of Mg2+. Borate-magnesium reaction is inhibited also by some polyvalent alcohols (glycerol) and sugars.

Efficiency of a novel non-Shine-Dalgarno and a Shine-Dalgarno consensus sequence to initiate translation in Escherichia coli of genes with different downstream box composition.

The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.

A polylinker-derived sequence, PL, highly increased translation efficiency in Escherichia coli.

Pokeweed (Phytolacca americana) antiviral protein (PAP) is a highly specific ribosome-inactivating glycosidase. The PAP gene was isolated and cloned in an expression vector containing a polylinker-derived sequence (PL) but devoid of a Shine-Dalgarno (SD) sequence. Surprisingly, E. coli cells transformed with this vector produced over twice the amount of PAP than that with the consensus SD sequence. Computer analysis of the 5' terminal region of PAP mRNA revealed a nucleotide sequence (ACCUACUCGAGUUAG) which was complementary to two domains in 16S rRNA. The heptanucleotide ACCUACU (box I) is complementary to nucleotides 1434-1440 and the GAGUUAG (box II) to nucleotides 507-513 in 16S rRNA of E. coli. To examine the role of this sequence in the translation of PAP mRNA, single or both boxes were mutated and the protein yield was measured. Mutation of box I and of box II resulted in a 2.7 and 5.3 fold decrease in protein yield respectively, indicating that the PAP gene expression was dependent on the presence of both boxes. To investigate whether PL also increases expression of other genes, human calcitonin monomeric and tetrameric genes were used as reporters. It was found that the expression level was doubled compared to that by SD. These results demonstrate that the PL is an efficient translational initiator and may be used for high level expression of certain genes in E. coli. The possible mechanisms for the high level expression are discussed.

The low expression level of pokeweed antiviral protein (PAP) gene in Escherichia coli by the inducible lac promoter is due to inefficient transcription and translation and not to the toxicity of the PAP.

Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosidase which inactivates both eukaryotic and prokaryotic ribosomes. Due to this activity the wild-type PAP gene encoding mature protein has not so far been expressed in Escherichia coli. In spite of the ribosome impairing activity of the pre-PAP (containing two signal peptides at both termini) on bacterial ribosomes in vitro, the full-length PAP gene has been expressed successfully, although at a low level in E. coli under an inducible lac promoter. In this study we show that the full-length PAP gene, but not the PAP gene devoid of the N-terminal signal peptide codons, can be expressed constitutively in E. coli cells to produce a much higher yield as compared with the inducible expression. The full-length PAP is biologically active and it accumulates as inclusion bodies in bacterial cytoplasm. RNA analysis together with protein measurements show that the PAP gene is poorly transcribed and the PAP mRNA is poorly translated when a lac operator sequence is placed in front of the Shine/Dalgarno (SD) sequence. Nucleotide folding analysis of the 5' untranslated mRNA revealed that the SD sequence in the presence of a lac operator is involved in a stable secondary structure, whereas it is more relaxed in the mRNA transcribed from the constitutive vector. These results provide evidence that the low expression level of full-length PAP gene is due to inefficient transcription and translation but not to the toxicity of the expressed PAP.

Self-cleaving circular RNA associated with rice yellow mottle virus is the smallest viroid-like RNA.

We report the sequence, structural features, and self-cleaving activity of the small circular RNA (sc-RNA) associated with rice yellow mottle sobemovirus (RYMV). At 220 nucleotides, the RYMV sc-RNA represents the smallest naturally occurring viroid-like RNA currently documented in the literature. It is similar to other circular satellite RNAs (sat-RNAs) and viroids in being G-C-rich with a high level of self-complementarity. The predicted native structure is essentially a rod with one branched terminus. A region of the RYMV sc-RNA, constituting 24% of the sequence, exhibits 89% identity to the sat-RNA associated with the Australasian isolates of lucerne transient streak sobemovirus. This region is also structurally similar in all three RNAs in that it forms the left terminus of each rod. Dimeric runoff transcripts of cloned RYMV sc-RNA undergo efficient autocatalytic in vitro cleavage in the (+) but not the (-) polarity. Analysis of the (+) sequence indicates the presence of a hammerhead ribozyme resembling that of carnation small retroviroid-like RNA and the genomic satellite transcript of newt. Inefficient cleavage of (+) monomeric transcripts, and a short stem III in the hammerhead, are features consistent with a double-hammerhead mode of self-cleavage. The presence of sat-RNA and retroviroid-like structures within a single RNA suggests a possible role for the RYMV sc-RNA as an evolutionary intermediate between these subviral RNAs.

Effect of N-terminal deletions on the activity of pokeweed antiviral protein expressed in E. coli.

Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A4324 in 28S rRNA and A2660 in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes. Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli. In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E. coli; and 2) the location of the putative enzymatic active site of PAP, 5'-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site. The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E. coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E. coli cells. Results showed that the native full-length PAP gene was highly expressed and was not toxic to E. coli cells although in vitro ribosome inactivating activity assay indicated it was active. However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E. coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAP delta107). Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones. These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E. coli cells. However, it is activated by at least seven codon deletions at the N-terminus. Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained. But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E. coli. Because deletion of Tyr94 and Val95, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.

Expression of the coat protein of potato virus X from a dicistronic mRNA in transgenic potato plants.

Transgenic potato plants were generated which express a dicistronic mRNA corresponding to the 8 kDa protein and coat protein (CP) genes of potato virus X (PVX). Northern blot analysis of RNA isolated from these plants indicated that both the 8 kDa protein and the CP are translated from this dicistronic mRNA. Expression of both of these proteins in transgenic plants was demonstrated by Western blot analysis, and suggests that translation of CP takes place either by initiation by internal entry of ribosomes or by a termination/reinitiation mechanism. CP was detected in protoplasts electroporated with RNA transcripts of the dicistronic construct in the presence or absence of a stable hairpin structure at the 5' terminus, suggesting that the former model is more likely. Similar results were obtained when a DNA fragment containing the PVX CP leader sequence was placed between two reporter genes in the transient assay system. These results suggest that potexvirus CP expression may take place from the genomic RNA as well as from the subgenomic RNA.

Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit.

A non-Shine-Dalgamo translational initiator is identified in Escherichia coli. The nucleotide sequence ACCUACUCGAGUUAG, designated as PL, is capable of initiating translation of pokeweed antiviral protein (PAP) and human calcitonin (hCT) mRNAs in E. coli cells. The yield of recombinant protein was double that obtained with the consensus Shine-Dalgarno-sequence-(SD)-driven translation. The PL sequence is composed of two heptanucleotides (ACCUACU, box I and GAGUUAG, box II) which are complementary to nucleotides 1434-1440 and 507-513, respectively, in 16S rRNA. Mutational analysis shows that the translation initiation efficiency with either box alone is much lower than that obtained with the entire PL sequence, indicating that the boxes interact simultaneously with both complementary regions in 16S rRNA during the translation initiation step. Based on these results, we propose that the two widely separated regions 507-513 (part of helical domain 18) and 1434-1440 (belonging to helical domain 44) are organized in close proximity to each other and to the ribosome decoding center on the surface of the E. coli 30S ribosomal subunit.

Does the epsilon sequence of phage T7 function as an initiator for the translation of CAT mRNA in Escherichia coli?

Epsilon (epsilon) sequence [UUAACUUUA, complementary to nucleotides 458-466 of the 16S ribosomal RNA (rRNA)] which is naturally occurring at the 5'-untranslated leader of phage T7 gene 10 mRNA was originally described as a powerful translational enhancer in Escherichia coli. Recent studies with this sequence led to controversial conclusions about its translational initiation and enhancing capability. In this study different sequence derivatives of epsilon were constructed to evaluate its efficiency not only to enhance translation of the chloramphenicol acetyltransferase (CAT) mRNA in E. coli, but also to function as an independent initiator of translation. It was observed that the epsilon sequence in combination with the CAT natural Shine-Dalgarno (SDn) or the SD consensus sequences enhanced, as expected, the translation of CAT mRNA. The natural epsilon sequence without an SD sequence failed to initiate or enhance the translation of CAT mRNA. However, when the complementarity of epsilon to 16S rRNA was increased from 9 to 16 nucleotides, epsilon alone (without the SD sequence) became an independent translational initiator with an efficiency of about 80% that obtained with the SD consensus sequence.

Unusual effect of clusters of rare arginine (AGG) codons on the expression of human interferon alpha 1 gene in Escherichia coli.

The human interferon (hIFN alpha 1) gene contains 11 arginine (Arg) codons AGG or AGA, which are extremely rare for bacteria, four of which are organized in tandems. The two AGG tandems (corresponding to Arg12 Arg13 and Arg163 Arg164) are known to inhibit the translation of hIFN alpha 1 mRNA and therefore they are considered to be responsible for the poor expression of hIFN alpha 1 gene in bacterial cells. To study the effect of these two tandems on the expression of hIFN alpha 1 in E. coli, four new gene variants were designed to contain preferential Arg codons (CGT) substituted for the rare AGG codons in either the first, the second or both AGG tandems. We found that, whereas the yield of hIFN alpha 1 protein per cell remained unchanged, the level of hIFN alpha 1 mRNA decreased gradually (by a factor of two) with the consecutive substitution of the first, second and both AGG tandems. These results indicated, first, that the AGG clusters might have a stabilizing effect on the mRNA, and second, that mRNAs devoid of such clusters were translated at a higher rate in vivo. The protein products of the four genes (having the same amino acid sequence) showed different specific antiviral activity. The most active was the product of gene hIFN alpha 1(c) in which the second AGG tandem (corresponding to Arg163, Arg164) was preserved while the least active was the protein of gene hIFN alpha 1(d) (devoid of both AGG clusters). The role of the AGG tandems in folding of the gene product is discussed.

Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato.

Open reading frame 4 (ORF4) of the potato virus X (PVX) genome encodes an 8K protein which is a part of the "triple gene block" and is known to play a role in the cell-to-cell movement of the virus in infected plants. To locate the 8K protein and further elucidate the mechanism of cell-to-cell transport of PVX, antibodies were raised against the 8K protein and used to localize this protein in PVX-infected tobacco and in transgenic potato plants expressing the 8K protein both by subcellular fractionation and by immunolabeling with colloidal gold. The results indicated that the 8K protein was localized to the cell wall.

Comparative study on the effect of signal peptide codons and arginine codons on the expression of human interferon-alpha 1 gene in Escherichia coli.

Human interferon-alpha 1 (HuIFN-alpha 1) gene containing signal peptide codons is poorly expressed in bacteria, and this is explained by the presence of clusters of rare (AGG) arginine codons in its structure. In this study, we have constructed a series of modified HuIFN-alpha 1 genes to study the effect of both residual signal peptide codons and clusters of AGG codons on gene expression in Escherichia coli cells. Our results showed that substitution of preferential for rare arginine codons in two clusters did not affect the yield, whereas deletion of the signal peptide codons led to a 10-fold increase in the yield of recombinant protein. To understand the mechanism of interference of gene structure on the expression of the HuIFN-alpha 1 gene in vivo, both the level and stability of HuIFN-alpha 1 mRNA were measured. The amount of HuIFN mRNA increased almost five times on deletion of the signal peptide codons from HuIFN-alpha 1 gene constructs (containing AGG clusters or not). The stability of mRNA obtained from all gene constructs was shown to be the same (half-life of 60 +/- 5 secs), indicating that the signal peptide codons interfere with both the efficiency of transcription of the HuIFN-alpha 1 gene and translation of its mRNA.

A second putative mRNA binding site on the Escherichia coli ribosome.

Translation in bacteria is initiated by a base-pairing interaction between the extreme 3'-end of the small-subunit rRNA and a purine-rich domain (Shine-Dalgarno (SD) sequence) preceding the initiation codon at the 5'-end of most bacterial mRNAs. Here, we describe the identification of a second functional and alternative site on the Escherichia coli ribosome which is capable of interacting with mRNA devoid of SD sequences and initiate the translation. This site is localized between nt 1340 and 1360 of the 16S rRNA in E. coli and is complementary to the untranslated region at the 5'-end of tobacco mosaic virus RNA (omega sequence).

Domains in human interferon alpha-1 gene containing tandems of arginine codons AGG play the role of translational initiators in E. coli.

The AGG and AGA are the least used arginine codons in E. coli but they are the most preferable ones in eukaryotes. The low expression of some eucaryotic genes (such as human alpha-1 interferon gene) which contain clusters of AGG codons is explained either by the limited pool of the tRNA(AGG) (Varenne and Lazdunski, 1986) or by the competition of these clusters with the Shine-Dalgarno (SD) sequence (Ivanov et al., 1992). The aim of the present study is to demonstrate the in vivo capacity of AGG tandems to bind to bacterial ribosomes. The two tandems of AGG codons (Arg12 Arg13 and Arg163 Arg164) of hIF alpha 1 with their surrounding nucleotides were cloned in a bacterial expression plasmid containing a strong promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) devoid of a ribosome binding site. The results obtained showed that both AGG tandems initiated translation of the CAT mRNA with an efficiency equal to that of the consensus SD sequence and several fold higher than the native SD sequence of the CAT gene.

Transgenic tobacco plants expressing a truncated form of the PAMV capsid protein (CP) gene show CP-mediated resistance to potato aucuba mosaic virus.

Four constructs of the potato aucuba mosaic virus (PAMV) coat protein (CP) gene were engineered for expression in tobacco plants: The full-length CP sequence (FL CP), two truncated forms--one at the N terminus (46 amino acid residues are deleted) (NT138), one in the conserved core portion (86 amino acids deleted) (CT258) of the gene--and an antisense RNA construct. These constructs were introduced into tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation. The plants transformed with the NT138 and FL CP constructs produced the mRNAs and proteins from the respective transgene. Transformants with the CT258 construct produced the transgenic mRNA, but the modified CP was not detected in the 20 different transformants tested. Transgenic R0 and R1 tobacco plants expressing the full-length, CT258, and the antisense constructs exhibited protection to PAMV infection and a delay in symptom development when inoculated with 0.1 and 0.5 microgram/ml of purified PAMV. Transgenic plants expressing the NT138 construct did not confer any detectable protection to PAMV infection. These results suggest that an engineered coat protein mediated resistance (CPMR) can be obtained from a CP gene truncated in its core region. The role of the N-terminal domain of the CP in the CPMR of PAMV and the implication of either the RNA or the protein in the protection is discussed.