Identification and characterization of E.coli ribosomal binding sites by free energy computation.

Sequences upstream from translational initiation sites of different E.coli genes show various degrees of complementarity to the Shine-Dalgarno (SD) sequence at the 3' end of the 16S rRNA. We propose a quantitative measure for the SD region on the mRNA, that reflects its degree of complementarity to the rRNA. This measure is based on the stability of the rRNA-mRNA duplex as established by free energy computations. The free energy calculations are based on the same principles that are used for folding a single RNA molecule, and are executed by similar algorithms. Bulges and internal loops in the rRNA and mRNA are allowed. The mRNA string with maximum free energy gain upon binding to the rRNA is selected as the most favorable SD sequence of a gene. The free energy value that represents the SD region provides a quantitative measure that can be used for comparing SD sequences of different genes. The distribution of this measure in more than 1000 E.coli genes is presented and discussed.     

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.     

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.     

Efficient production of a human tumour necrosis factor in Escherichia coli

To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell.     

Isolation and sequence analysis of rpoH genes encoding sigma 32 homologs from gram negative bacteria: conserved mRNA and protein segments for heat shock regulation.

The rpoH genes encoding homologs of Escherichia coli sigma 32 (heat shock sigma factor) were isolated and sequenced from five gram negative proteobacteria (gamma or alpha subgroup): Enterobacter cloacae (gamma), Serratia marcescens (gamma), Proteus mirabilis (gamma), Agrobacterium tumefaciens (alpha) and Zymomonas mobilis (alpha). Comparison of these and three known genes from E.coli (gamma), Citrobacter freundii (gamma) and Pseudomonas aeruginosa (gamma) revealed marked similarities that should reflect conserved function and regulation of sigma 32 in the heat shock response. Both the sequence complementary to part of 16S rRNA (the 'downstream box') and a predicted mRNA secondary structure similar to those involved in translational control of sigma 32 in E.coli were found for the rpoH genes from the gamma, but not the alpha, subgroup, despite considerable divergence in nucleotide sequence. Moreover, a stretch of nine amino acid residues Q(R/K)(K/R)LFFNLR, designated the 'RpoH box', was absolutely conserved among all sigma 32 homologs, but absent in other sigma factors; this sequence overlapped with the segment of polypeptide thought to be involved in DnaK/DnaJ chaperone-mediated negative control of synthesis and stability of sigma 32. In addition, a putative sigma E (sigma 24)-specific promoter was found in front of all rpoH genes from the gamma, but not alpha, subgroup. These results suggest that the regulatory mechanisms, as well as the function, of the heat shock response known in E.coli are very well conserved among the gamma subgroup and partially conserved among the alpha proteobacteria.     

Use of a transactive regulatory mutant of Dictyostelium discoideum in a eucaryotic expression system.

The discoidin proteins of Dictyostelium discoideum are highly expressed during development. The Disc I gamma promoter allows the regulation of heterologous protein expression by experimental conditions. We report conditions under which the promoter activity is efficiently repressed during growth in the wildtype strain AX2. In addition we show that a mutant which overexpresses the discoidins also overexpresses the reporter genes beta-galactosidase, luciferase and CAT 10- to 100-fold when these are placed under the control of a Disc I gamma promoter. This system may be generally useful for the overexpression of genes in Dictyostelium, both for functional studies in vivo and for the production of heterologous proteins for purification.     

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.     

The S(MK) box is a new SAM-binding RNA for translational regulation of SAM synthetase

We have identified the S(MK) box as a conserved RNA motif in the 5' untranslated leader region of metK (SAM synthetase) genes in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element bound SAM in vitro, and binding of SAM caused an RNA structural rearrangement that resulted in sequestration of the Shine-Dalgarno (SD) sequence. Mutations that disrupted pairing between the SD region and a sequence complementary to the SD blocked SAM binding, whereas compensatory mutations that restored pairing restored SAM binding. The Enterococcus faecalis S(MK) box conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated, and mutations that blocked SAM binding resulted in loss of repression, demonstrating that the S(MK) box is functional in vivo. The S(MK) box therefore represents a new SAM-binding riboswitch distinct from the previously identified S box RNAs.     

Computational design of orthogonal ribosomes

Orthogonal ribosomes (o-ribosomes), also known as specialized ribosomes, are able to selectively translate mRNA not recognized by host ribosomes. As a result, they are powerful tools for investigating translational regulation and probing ribosome structure. To date, efforts directed towards engineering o-ribosomes have involved random mutagenesis-based approaches. As an alternative, we present here a computational method for rationally designing o-ribosomes in bacteria. Working under the assumption that base-pair interactions between the 16S rRNA and mRNA serve as the primary mode for ribosome binding and translational initiation, the algorithm enumerates all possible extended recognition sequences for 16S rRNA and then chooses those candidates that: (i) have a similar binding strength to their target mRNA as the canonical, wild-type ribosome/mRNA pair; (ii) do not bind mRNA with the wild-type, canonical Shine-Dalgarno (SD) sequence and (iii) minimally interact with host mRNA irrespective of whether a recognizable SD sequence is present. In order to test the algorithm, we experimentally characterized a number of computationally designed o-ribosomes in Escherichia coli.     

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.     

Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli

Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3′ end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions ?9 to ?5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

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.     

Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA

We identified a short RNA fragment, complementary to the Escherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.     

Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.     

Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli

Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3′ end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions −9 to −5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs. Received: 8 July 1997 / Accepted: 9 September 1997     

Inability of Agrobacterium tumefaciens ribosomes to translate in vivo mRNAs containing non-Shine-Dalgarno translational initiators

Numerous data accumulated during the last decade have shown that the Shine-Dalgarno (SD) sequence is not a unique initiator of translation for Escherichia coli. Several other sequences, mostly of viral origin, have demonstrated their capability of either enhancing or initiating translation in vivo. A phage T7 gene 10 sequence, called "epsilon" (epsilon), has shown its high enhancing activity on translation in both Escherichia coli and Agrobacterium tumefaciens cells. In this study the epsilon, together with three other nucleotide sequences derived from the 5' non-translated regions of tobacco mosaic virus (TMV), papaya mosaic virus (PMV) and clover yellow mosaic virus (CYMV) RNAs are tested for translation initiation activity in A. tumefaciens cells. The obtained results indicate that none of them was capable of initiating translation in vivo of chloramphenicol acetyltransferase (CAT) mRNA. To determine whether their inactivity was related with structural differences in the ribosomal protein S1, the rpsA gene (coding for S1 protein in E. coli) was co-expressed in A. tumefaciens together with the cat gene placed under the translational control of the above sequences. Our results showed that the rpsA gene product did not make any of the four viral enhancers active in A. tumefaciens cells. The inability of A. tumefaciens ribosomes to translate mRNAs devoid of SD sequences indicates for a substantial difference in the ribosome structure of the two Gram negative bacteria E. coli and A. tumefaciens.