RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.     

Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli

Translational efficiency in Escherichia coli is known to be strongly influenced by the secondary structure around the ribosome‐binding site and the initiation codon in the translational‐initiation region of the mRNA. Several quantitative studies have reported that translational efficiency is attributable to effects on ribosome accessibility predominantly caused by the secondary structure surrounding the ribosome‐binding site. However, the influence of mRNA secondary structure around regions downstream of the initiation codon on translational efficiency after ribosome‐binding step has not been quantitatively studied. Here, we quantitatively analyzed the relationship between secondary structure of mRNA surrounding the region downstream of the initiation codon, referred to as the downstream region (DR), and protein expression levels. Modified hairpin structures containing the initiation codon were constructed by site‐directed mutagenesis, and their effects on expression were analyzed in vivo. The minimal folding free energy (ΔG) of a local hairpin structure was found to be linearly correlated with the relative expression level over a range of fourfold change. These results demonstrate that expression level can be quantitatively controlled by changing the stability of the secondary structure surrounding the DR. Biotechnol. Bioeng. 2009; 104: 611–616 © 2009 Wiley Periodicals, Inc.     

Expression of gonococcal protein II in Escherichia coli by translational fusion

A protein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of beta-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II.     

Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli

When expressing several eukaryotic genes in Escherichia coli, we observed N-terminally truncated proteins which were attributed to translation initiation at downstream AUG codons. These AUG codons are located between 4 and 20 nucleotides 3' from sequences resembling bacterial SD elements. Although the presence of such downstream SD sequences is not sufficient for downstream initiation to occur, in two cases their removal abolishes synthesis of the truncated proteins. In one construct, a potential hairpin-loop structure is likely to inhibit translation initiation at the correct site and favor downstream initiation.     

Recognition of messenger RNA during translational initiation in Escherichia coli

The structural aspects of recognition by E. coli ribosomes of translational initiation regions on homologous messenger RNAs have been reviewed. Also discussed is the location of initiation region on mRNA, its confines, typical nucleotide sequences responsible for initiation signal, and the influence of RNA macrostructure on protein synthesis initiation. Most of the published DNA nucleotide sequences surrounding the start of various E. coli genes and those of its phages have been collected.     

Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.     

Independent and coupled translational initiation of atp genes in Escherichia coli: experiments using chromosomal and plasmid-borne lacZ fusions

The translational initiation rates directed by the translational initiation regions (TIRs) of the atpB, atpH, atpA and atpG genes of Escherichia coli were investigated using lacZ fusions present on plasmids as well as integrated into the chromosome. This was the first investigation of the translational efficiency of the atpB gene, whose unfused product (subunit a) can be toxic to the cell. The specific mRNA levels, rates of in vivo protein synthesis and beta-galactosidase activities encoded by the atp::lacZ fusions were compared in order to obtain valid estimates of relative translation rates. The results indicate that in the E. coli atp operon, translation directed by the atpB, atpH and atpG TIRs is less efficient than that directed by the atpA TIR, and are thus consistent with earlier measurements of direct atp gene expression. Initiation is, however, to differing extents, controlled by coupling to the translation of upstream neighbours. There is particularly tight coupling between atpH and atpA. Increasing the distance between these two genes whilst maintaining the original atpA TIR structure decreased the degree of coupling. The influence of manipulations of the atpG TIR structure upon translational efficiency was quantitatively more pronounced when the atpG fusions were present as a single copy per chromosome. This is likely to be related to the mRNA binding characteristics of 30S ribosomal subunits and/or to the influence of other (trans-acting) factors. The control of independent and coupled initiation at the atp TIRs is discussed in relation to mRNA structure and possible cis and trans regulatory phenomena.     

Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.     

RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging.     

The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5' untranslated leader

Translational efficiency of an AUG, CUG, GUG, or UUG initiation codon was measured for the naturally leaderless cI mRNA from bacteriophage lambda. In a cI-lacZ translational fusion, only AUG supported a high level of expression; GUG supported a low level of expression, while UUG and CUG expression was barely above background levels. Addition of an untranslated lac leader and Shine-Dalgarno sequence to cI increased expression but still showed a dependence on an AUG for maximum expression. cI-lacZ mRNA with an AUG initiation codon showed a greater in vitro ribosome binding strength and a higher level of full-length in vivo mRNA, suggesting that the initiation codon is an important determinant of ribosome binding strength and translational efficiency for mRNA with or without the 5' untranslated leader.     

Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency.

The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes. Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions. However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position. The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization.     

Inferring rules of Escherichia coli translational efficiency using an artificial neural network

Although the machinery for translation initiation in Escherichia coli is very complicated, the translational efficiency has been reported to be predictable from upstream oligonucleotide sequences. Conventional models have difficulties in their generalization ability and prediction nonlinearity and in their ability to deal with a variety of input attributions. To address these issues, we employed structural learning by artificial neural networks to infer general rules for translational efficiency. The correlation between translational activities measured by biological experiments and those predicted by our method in the test data was significant (r=0.78), and our method uncovered underlying rules of translational activities and sequence patterns from the obtained skeleton structure. The significant rules for predicting translational efficiency were (1) G- and A-rich oligonucleotide sequences, resembling the Shine-Dalgarno sequence, at positions -10 to -7; (2) first base A in the initiation codon; (3) transport/binding or amino acid metabolism gene function; (4) high binding energy between mRNA and 16S rRNA at positions -15 to -5. An additional inferred novel rule was that C at position -1 increases translational efficiency. When our model was applied to the entire genomic sequence of E. coli, translational activities of genes for metabolism and translational were significantly high.