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.     

A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli

We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S. (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."     

Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs.

The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.     

Cooperative effects by the initiation codon and its flanking regions on translation initiation

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.     

Translational enhancement by an element downstream of the initiation codon in Escherichia coli

The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addition to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD.     

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region promotes accumulation of NPTII up to ~16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield (~23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.     

Translation enhancement by optimized downstream box sequences in <Emphasis Type="Italic">Escherichia coli </Emphasis>and<Emphasis Type="Italic"> Mycobacterium smegmatis</Emphasis>

The effect on translation of downstream box sequences optimized for binding to Mycobacterium smegmatis and Escherichia coli 16S rRNA in the absence of a Shine--Dalgarno (SD) region was investigated. The relative translational efficiency of each construct in either M. smegmatis or E. coli was determined. Eradication of the SD region in the absence of a downstream box abolished the translation activity. In contrast, optimized downstream box constructs resulted in a 13- and 18-fold increase in protein synthesis, relative to non-optimized DB controls in E. coli and M. smegmatis, respectively.     

Codon and base biases after the initiation codon of the open reading frames in the Escherichia coli genome and their influence on the translation efficiency

Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed. Characteristic base biases were observed after the initiation codon and before the termination codon. We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E. coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems. In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant. Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected. These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.     

Essential elements in the coding region of mRNA for translation of ColE2 Rep protein

Translation initiation of mRNA encoding the plasmid-specified initiator protein (Rep) required for initiation of the ColE2 plasmid DNA replication is fairly efficient in Escherichia coli despite the absence of a canonical Shine-Dalgarno sequence. Although a GA cluster sequence exists upstream the initiation codon, its activity as the SD sequence has been shown to be very inefficient. Deletion analyses have shown that there are sequences important for the Rep translation in the regions upstream the GA cluster sequence and downstream the initiation codon. To further define regions important for translation of the Rep mRNA, a set of the ColE2 rep genes bearing single-base substitution mutations in the coding region near the initiation codon was generated and their translation activities examined. We showed that translation of the Rep mRNA was reduced by some of these mutations in a region ranging at least 70 nucleotides from the initiation codon in the coding region, indicating the presence of translation enhancer(s) outside the translation initiation region which is covered by the ribosome bound to the initiation codon. Some of them seem to be essential and specific for translation of the ColE2 Rep mRNA due to the absence of a canonical SD sequence.     

Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts.

Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA-lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5'-untranslated region of the mRNA are required for translation. Two of them are complementary to the 3'-terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans-acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3'-end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans-acting factor(s).     

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.     

Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis.

Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support. The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2. The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates. The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence. There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences. Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.     

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.     

Broad-specificity mRNA-rRNA complementarity in efficient protein translation

Studies of synthetic, well-defined biomolecular systems can elucidate inherent capabilities that may be difficult to uncover in a native biological context. Here, we used a minimal, reconstituted translation system from Escherichia coli to identify efficient ribosome binding sites (RBSs) in an unbiased, high-throughput manner. We applied ribosome display, a powerful in vitro selection method, to enrich only those mRNA sequences which could direct rapid protein translation. In addition to canonical Shine-Dalgarno (SD) motifs, we unexpectedly recovered highly efficient cytosine-rich (C-rich) sequences that exhibit unmistakable complementarity to the 16S rRNA of the small subunit of the ribosome, indicating that broad-specificity base-pairing may be an inherent, general mechanism for efficient translation. Furthermore, given the conservation of ribosomal structure and function across species, the broader relevance of C-rich RBS sequences identified through our in vitro evolution approach is supported by multiple, diverse examples in nature, including C-rich RBSs in several bacteriophage and plants, a poly-C consensus before the start codon in a lower eukaryote, and Kozak-like sequences in vertebrates.     

Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.     

A novel translation initiation region from Mycoplasma genitalium that functions in Escherichia coli.

The tuf gene of Mycoplasma genitalium uses a signal other than a Shine-Dalgarno sequence to promote translation initiation. We have inserted the translation initiation region of this gene in front of the Escherichia coli lacZ gene and shown that it is recognized by the translational machinery of E. coli; the signal operates in vivo at roughly the same efficiency as a synthetic Shine-Dalgarno sequence. The M. genitalium sequence was also used to replace the native translation initiation region of the cat gene. When assayed in E. coli, the M. genitalium sequence is equivalent to a Shine-Dalgarno sequence in stimulating translation of this mRNA also. Site-directed mutagenesis enabled us to identify some of the bases that comprise the functional sequence. We propose that the sequence UUAACAACAU functions as a ribosome binding site by annealing to nucleotides 1082-1093 of the E. coli 16S rRNA. The activity of this sequence is enhanced when it is present in the loop of a stem-and-loop structure. Additional sequences both upstream and downstream of the initiation codon are also involved, but their role has not been elucidated.     

A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.     

Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs

Many of the chloroplast mRNAs possess Shine-Dalgarno (SD)-like sequences (typically GGAGG) in the 5'-untranslated regions, but the position is highly variable. Using a homologous in vitro translation system, we assessed the role for translation of SD-like sequences in four tobacco chloroplast mRNAs. The rbcL mRNA has a typical SD-like sequence at a position similar to the conserved position (-12 to -4 with respect to the start codon) observed in E. coli, and this sequence was found to be essential for translation. This was also the case for the atpE mRNA. However, SD-like sequences in the rps12 mRNA and in the petB mRNA is located far from (-44 to -42) and too close to (-5 to -2) the initiation codon, respectively, and these sequences were not essential for translation. These results indicate that functional SD-like sequences are located around 10 nucleotides upstream from the translational start codon. Competition assays confirmed that a functional SD-like sequence interacts with the 3' terminus of chloroplast 16S rRNA.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.