Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens

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Highlights

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    Several viral and chloroplastic 5′ translational contexts were examined for translational efficiency in A. tumefaciens.

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    The T7 translational enhancer together with the ribosome binding site generated the highest expression in A. tumefaciens.

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    Among chloroplastic 5′ contexts, the large subunit of rubisco (rbcL) 58 base 5′ UTR exhibited the most robust expression.

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    Replacing the SD-like sequence or introducing mutations to the remainder of the rbcL 5′ UTR abrogated expression.

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    The 5′ UTR of rbcL sequence also functioned as a translational enhancer in plants.

Abstract

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5′ leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5′ UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5′ UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5′ leader region upstream of the gene of interest.

Introduction

Initiation of translation in Escherichia coli involves base pairing between a purine-rich Shine–Dalgarno (SD) domain at the 5′ untranslated region (5′ UTR) of mRNA, and the complementary anti-SD sequence at the 3′ end of 16S rRNA [1]. There are distinct sequence elements of the translation initiation region known to contribute to its efficiency [2]: the initiation codon, the Shine–Dalgarno (SD) sequence [3], [4] as well as regions upstream of the SD sequence and downstream of the initiation codon, described as enhancers of translation [5]. The distance between the SD sequence and the initiation triplet has a marked effect on the efficiency of translation [6]. The 6-nucleotide consensus SD AGGAGG core sequence causes the highest level of protein synthesis.

Chloroplasts have their own translation system, which exhibits strong homologies to that of prokaryotes. This is consistent with the presence of a Shine–Dalgarno (SD) sequence (GGAGG) located within 12 nucleotides of the AUG initiation codon of many plastid genes [7]. Moreover, the sequence near the 3′ end of the plastid 16S rRNA contains a highly conserved polypyrimidine-rich region (CCUCC) complementary to the SD sequence as in bacteria. Over 90% of higher plant chloroplast genes encoding polypeptides possess an upstream sequence similar to the bacterial SD sequence. Spacing of these chloroplast SD-like sequences is less conserved, ranging from −2 to −29 nucleotides [8]. Translation of several chloroplast mRNAs is also regulated in response to light as well as to some nuclear-encoded factors. In this regard, it is interesting to study how well chloroplastic translational machinery function in Eubacteria such as E. coli and Agrobacterium tumefaciens.

The transfer of T-DNA from Agrobacterium into the plant genome represents a natural horizontal gene transfer across kingdom barriers and implicates a closer evolutionary relationship between Agrobacterium and plants than between any other Eubacterial organism (such as E. coli) and plants. The aim of the present study is to investigate the sequence determinants responsible for efficient translation in A. tumefaciens, which on the one hand is highly similar to E. coli in terms of its dependency on the SD sequence for the translation, while on the other hand is also mechanistically similar to chloroplast genes such as the large subunit of the Rubisco in its dependence on the 5′ upstream control region. Also, the essential molecular determinants for the design of an ideal Agrobacterial expression vector are considered.

Section snippets

Construction of GFP expression plasmids

The binary vector pCAMBIA1300 (CAMBIA, Canberra, Australia) was used in this study. A 35S: sGFP:NOS expression cassette (GenBank EF546437) of size 1.9-kbp was subcloned into this vector through HindIII and EcoRI sites and designated pC-GFP (Fig. 1A). To create the pCTCR-GFP construct, the translation control region (TCR) [9], comprised of 58 nucleotides of 5′ UTR and 45 nucleotides from the N-terminal coding region of the rbcL gene were synthesized and cloned into pUC57 plasmid (Bio Basic Inc.)

Results and Discussion

GFP expression in ten pCAMBIA constructs (Fig. 1B) containing different translation initiation contexts upstream of the GFP gene was monitored by confocal microscopy and Western blot analysis, after transformation of A. tumefaciens (JV3101 strain) with the respective constructs. All constructs uniformly contained the CaMV 35S promoter and the enhanced GFP gene followed by the 3′ NOS terminator. This produces the same GFP transcript levels for all the constructs. The only difference between the

Acknowledgment

We thank both NSERC - Canada and OGS for each partially providing funding for T.A.

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