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.     

Escherichia coli mRNAs with strong Shine/Dalgarno sequences also contain 5' end sequences complementary to domain # 17 on the 16S ribosomal RNA

A well-established feature of the translation initiation region, which attracts the ribosomes to the prokaryotic mRNAs, is a purine rich area called Shine/Dalgarno sequence (SD). There are examples of various other sequences, which despite having no similarity to an SD sequence are capable of enhancing and/or initiating translation. The mechanisms by which these sequences affect translation remain unclear, but a base pairing between mRNA and 16S ribosomal RNA (rRNA) is proposed to be the likely mechanism. In this study, using a computational approach, we identified a non-SD signal found specifically in the translation initiation regions of Escherichia coli mRNAs, which contain super strong SD sequences. Nine of the 11 E. coli translation initiation regions, which were previously identified for having super strong SD sequences, also contained six or more nucleotides complementary to box-17 on the 16S rRNA (nucleotides 418-554). Mutational analyses of those initiation sequences indicated that when complementarity to box-17 was eliminated, the efficiency of the examined sequences to mediate the translation of chloramphenicol acetyltransferase (CAT) mRNA was reduced. The results suggest that mRNA sequences with complementarity to box-17 of 16S rRNA may function as enhancers for translation in E. coli.     

Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF-lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem-loop with that of Salmonella enterica serovar Typhimurium or a stem-loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon-rRNA base pairing does not occur.     

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.     

Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence.

Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol. Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA. Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence. Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame. Codons 2 through 5 specify Val-Lys-Thr-Asp. Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold. Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure. We suggest that the stall sequence may function in cat induction at two levels. First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W. W. Mulbry, N. P. Ambulos, Jr., and P.S. Lovett, J. Bacteriol. 171:5322-5324, 1989). Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis. This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both. The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E. coli.     

An additional ribosome-binding site on mRNA of highly expressed genes and a bifunctional site on the colicin fragment of 16S rRNA from Escherichia coli: important determinants of the efficiency of translation-initiation.

For various genes of E. coli, three regions (-55 to -1; -35 to -1; -21 to -1) 5' to AUG codon on mRNA were searched for sites of interaction with colicin fragment of 16S rRNA. The detailed sequence comparison points out that apart from Shine-Dalgarno base pairing, an additional ribosome-binding site, a subsequence of 5'-UGAUCC-3' invariably exists in mRNA for highly expressed genes. Poorly expressed genes appear to be controlled by only Shine-Dalgarno base pairing. The analysis indicates that in the initiator region, the -55 to -1 region contains the signal which decides the efficiency of the translation-initiation. The site on 16S rRNA, 5'-GGAUCA-3' at position 1529, that can base pair to the above site, has a recognition site on 23S rRNA at position 2390. In the light of the conserved nature and accessibility of these sites, it is proposed that the site on 16S rRNA plays a bifunctional role--initially it binds to mRNA from highly expressed genes to form a stable 30S initiation complex, and upon association with 50S subunit it exchanges base pairing with 23S rRNA, thus leaving the site on mRNA free.     

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.     

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.     

The 3'' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.     

Inability of Prevotella bryantii to form a functional Shine-Dalgarno interaction reflects unique evolution of ribosome binding sites in Bacteroidetes

The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5' untranslated mRNA regions (5'-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5'-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5'-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5'-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.     

Influences on gene expression in vivo by a Shine-Dalgarno sequence

The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3' to 5' ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD+ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.     

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.     

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 of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.     

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.