Efficiency of the 5′‐terminal sequence (Ω) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli

Abstract

Recent studies have demonstrated that the 5' leader (Q sequence) of tobacco mosaic virus RNA has a certain enhancing capacity for translation of mRNA in both prokaryotes and eukaryotes. In order to estimate the efficiency of 52 to initiate translation of mRNA in Escherichia coli, in comparison to the Shine-Dalgarno (S/D) sequence, we have inserted eight different eukaryotic genes into two types of E. coli expression vectors containing one constitutive promoter (Pl) but different translationinitiation sites (S/D or Q A 3 sequence, respectively). The efficiency of transcription and translation in vivo was evaluated for these vectors by measuring the yield of protein and both the level and stability of mRNA. We report that substitution of Q A 3 for S/D decreases the yield of expressed protein 4-1900-fold and the content of gene-specific mRNA is decreased by about sevenfold. However, in comparison with the S/D sequence, the level of protein expressed under the translational control of Q A 3 is less sensitive to changes in the 5' coding region. We also report that the 52 sequence contains a region of 10-12 nucleotides complementary to the small ribosomal subunit RNA (rRNA) of E. coli, Eikenella corrodens and Xenopus faevis, and to the rRNA of the (small ribosomal) subunit of Oryza sativa.It has been shown that the 5' untranslated region of the tobacco mosaic virus (TMV) RNA (52 sequence) enhances translation of mRNA in vivo and in vitro in both eukaryotes and prokaryotes [l -61. Gallie et al.[5] constructed a series of mutant sequence derivatives of the native Q sequence and studied their ability to enhance translation of two mRNA: pglucuronidase and chloramphenicol acetyltransferase in tobacco mesophyll protoplasts, Xenopus laevis oocytes and Escherichia roli. They found that deletion of the 25-base poly(CAA) sequence from the middle part of Q increased its enhancing effect in E. roli but not in eukaryotic cells. The efficiency of this sequence (designated QA3) to initiate translation in several types of bacteria has been studied, and the conclusion was drawn that Q is a sequence functionally equivalent to the Shine-Dalgarno (S/D) sequence [6].Unlike the native S/D sequence having the consensus AAGGAGGT [7, 81, the 5' untranslated region of the TMV RNA is devoid of G and is therefore unable to interact with the 3' end of 16s rRNA. This feature allows speculation that the 52 sequence initiates translation in bacteria by alternative mechanisms of binding of mRNA to the ribosomes.Several studies have shown that the efficiency of expression of a specific gene depends very much on the AUG-codonCorrespondence to M. G. AbouHaidar,

Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in Escherichia coli

“…Features of the mRNA RBS contributing to the efficiency of ribosome binding and translation include the start codon (most frequently AUG in Escherichia coli) and the purine-rich Shine-Dalgarno (SD) sequence, located within the untranslated leader region (UTR), which base pairs with the anti-SD (ASD) sequence near the 3Ј end of 16S rRNA (11,20,29). In addition to the start codon and SD-ASD interaction, other sequence and structural motifs within mRNA have been suggested to influence ribosome binding and translation, and these motifs include mRNA secondary structure within the TIR (6, 7), specific translationenhancing sequences upstream (10,12,21,37) and downstream (9,19,26,32) of the start codon, upstream pyrimidinerich tracts (1,38,42), AU-rich sequences within the UTR (14,15), and downstream A-rich (3) and CA-rich tracts (16). In addition, an increasing number of non-SD-led genes (2) and genes encoding mRNA lacking a 5Ј-untranslated leader region (leaderless mRNA) (13,18) are being identified, raising the potential that there are novel sequence and structural motifs within the coding sequence that contribute to the formation of translation initiation complexes.…”

Investigating the in vivo activity of the DeaD protein using protein–protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs

“…The filters were then treated with a secondary (anti-rabbit) IgG conjugated with alkaline phosphatase. The relative content and half-life of cat mRNA was determined by dot blot using a 33 Plabeled primer (GCCCATGGTGAAAACGGGG-GG) specific to cat mRNA in three independent experiments as described elsewhere [Ivanov et al, 1992]. The relative activity of each RNA spot was determined by a phosphor-imager (Biorad).…”

Non-Shine-Dalgarno Initiators of Translation Selected from Combinatorial DNA Libraries

“…The content of CAT mRNA in the transformed cells was measured by hybridization with 32 P-labeled oligonucleotides as described elsewhere [Ivanov et al, 1992]. The secondary structure of RNA was studied using the computer program PCGENE (IntelliGenetics).…”

“…The complex type of mRNA-ribosome interaction is illustrated as well by the existence of nucleotide sequences enhancing translation. Such sequences were found in several phage, viral and bacterial genes [Galie and Kado, 1989;Ivanov et al, 1992Ivanov et al, , 1995Kaloyanova et al, 1997;Loechel et al, 1991;Olins and Rangwala, 1989;Scherer et al, 1980;Shean and Gottesman, 1992;Sprengart et al, 1990;Ugarov et al, 1994;Wu and Janssen, 1997]. The first enhancer of translation (designed as ø sequence) that was active in both prokaryotic and eukaryotic cells [Galie and Kado, 1989] was identified at the 5)-nontranslated region of the tobacco mosaic virus (TMV) RNA.…”

“…It is noteworthy that unlike the purine-rich SD sequences, all bacterial enhancers of translation were poor in purines and particularly in guanine (G). Recent studies carried out in our laboratory have shown that besides their enhancing activity, some nucleotide sequences were also capable of independently (in the absence of SD sequence) initiating translation in Escherichia coli cells [Ivanov et al, 1992[Ivanov et al, , 1995Walz et al, 1976]. Min et al [1988] attempted to identify active translational initiators by screening a combinatorial library containing a randomized area at nucleotides -3 and -13 in an expression plasmid.…”