Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.     

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.     

Evidence against an Interaction between the mRNA downstream box and 16S rRNA in translation initiation

Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless lambda cI-lacZ reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973-8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393-1402, 2000), we conclude that the db does not base pair with the adb.     

The Shine and Dalgarno hypothesis for termination: the 3' terminus of the 16S rRNA of the Escherichia coli ribosome can be modified or base-paired with a complementary oligonucleotide without affecting termination in vitro

The occurrence of the nucleotides "...CCUUAOH" at the 3' terminus of the 16S rRNA of the small subunit of the Escherichia coli ribosome led to the suggestion that they may have a direct base pairing with the termination codon in the termination event of protein biosynthesis (Shine and Dalgarno 1974). We have examined this concept with two approaches, firstly using a 30S subunit whose 16S rRNA has been modified with a fluorescein moiety on the terminal adenosine together with the antibody against the moiety, and secondly with an oligonucleotide, UAAGG, complementary to the terminal pentanucleotide sequence of the rRNA. Collectively the data suggest that the nucleotides at the 3' terminus of 16S rRNA are not critically involved in base pairing during termination codon recognition.     

Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various prokaryotes.

MOTIVATION: It is well accepted that the 3' end of 16S rRNA is directly involved in prokaryotic translation initiation by pairing with the Shine-Dalgarno (SD) sequence, which is located in the ribosome-binding site of mRNA. According to Shine and Dalgarno, Escherichia coli 's 5' UTR has the pattern of 'AGGAGG' (SD sequence), which is complementary to the 3' end sequence of 16S rRNA. In this work, we systematically calculated free-energy values of the base pairing between the 3' end of 16S rRNA and the 5' UTR of mRNA, in order to analyze the base-pairing potentials in various prokaryotes. The free-energy values were then plotted over distances from the start codon to visualize the free-energy pattern of 5'UTRs. RESULTS: The average free-energy values fell sharply before the start codon in E. coli, which is consistent with the model that the 3' end of 16S rRNA base pairs with the SD sequence. Haemophilus influenzae, Bacillus subtilis and Helicobacter pylori show a similar pattern, suggesting that the organisms have basically the same mechanism of translation initiation as E. coli. Other eubacteria, such as Synechocystis PCC6803, Mycoplasma genitalium, Mycoplasma pneumoniae and Borrelia burgdorferi also show decreases in their free-energy values, although they are less evident. We also did the same analysis with a eukaryote genome as a control; no fall in free-energy values was observed between the 3' end of 18S rRNA and 5' UTRs of Saccharomyces cerevisiae, suggesting that this organism does not base pair in translation initiation. The three archaebacteria A. fulgidus, M. jannaschii and M. thermoautotrophicum show patterns similar to eubacteria, but not to S. cerevisiae, indicating that archaebacteria are closer to eubacteria than to eukaryotes with respect to the mechanism of translation initiation. From these observations, it appears that the shape of the curve produced by the algorithm can be used to predict the mechanism of translation initiation. AVAILABILITY: The C programs used in our analysis are available upon request.     

Initiation factor 3-induced structural changes in the 30 S ribosomal subunit and in complexes containing tRNA(f)(Met) and mRNA

Initiation factor 3 (IF3) acts to switch the decoding preference of the small ribosomal subunit from elongator to initiator tRNA. The effects of IF3 on the 30 S ribosomal subunit and on the 30 S.mRNA. tRNA(f)(Met) complex were determined by UV-induced RNA crosslinking. Three intramolecular crosslinks in the 16 S rRNA (of the 14 that were monitored by gel electrophoresis) are affected by IF3. These are the crosslinks between C1402 and C1501 within the decoding region, between C967xC1400 joining the end loop of a helix of 16 S rRNA domain III and the decoding region, and between U793 and G1517 joining the 790 end loop of 16 S rRNA domain II and the end loop of the terminal helix. These changes occur even in the 30 S.IF3 complex, indicating they are not mediated through tRNA(f)(Met) or mRNA. UV-induced crosslinks occur between 16 S rRNA position C1400 and tRNA(f)(Met) position U34, in tRNA(f)(Met) the nucleotide adjacent to the 5' anticodon nucleotide, and between 16 S rRNA position C1397 and the mRNA at positions +9 and +10 (where A of the initiator AUG codon is +1). The presence of IF3 reduces both of these crosslinks by twofold and fourfold, respectively. The binding site for IF3 involves the 790 region, some other parts of the 16 S rRNA domain II and the terminal stem/loop region. These are located in the front bottom part of the platform structure in the 30 S subunit, a short distance from the decoding region. The changes that occur in the decoding region, even in the absence of mRNA and tRNA, may be induced by IF3 from a short distance or could be caused by the second IF3 structural domain.     

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.     

Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli

Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3' terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.     

Control of translation initiation involves a factor-induced rearrangement of helix 44 of 16S ribosomal RNA

Initiation of translation involves recognition of the start codon by the initiator tRNA in the 30S subunit. To investigate the role of ribosomal RNA (rRNA) in this process, we isolated a number of 16S rRNA mutations that increase translation from the non-canonical start codon AUC. These mutations cluster to distinct regions that overlap remarkably well with previously identified class III protection sites and implicate both IF1 and IF3 in start codon selection. Two mutations map to the 790 loop and presumably act by inhibiting IF3 binding. Another cluster of mutations surrounds the conserved A1413∘G1487 base pair of helix 44 in a region known to be distorted by IF1 and IF3. Site-directed mutagenesis in this region confirmed that this factor-induced rearrangement of helix 44 helps regulate initiation fidelity. A third cluster of mutations maps to the neck of the 30S subunit, suggesting that the dynamics of the head domain influences translation initiation. In addition to identifying mutations that decrease fidelity, we found that many P-site mutations increase the stringency of start codon selection. These data provide evidence that the interaction between the initiator tRNA and the 30S P site is tuned to balance efficiency and accuracy during initiation.     

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.     

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.     

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and microevolution

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.     

Kinetic checkpoint at a late step in translation initiation

The translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA.     

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.     

Structural aspects of RbfA action during small ribosomal subunit assembly

Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.     

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.     

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.     

Computer analyses of complete genomes suggest that some archaebacteria employ both eukaryotic and eubacterial mechanisms in translation initiation

The translation initiation mechanism of archaebacteria is still not clearly understood. Our previous work showed that ATG triplets before start codons have been strongly depleted in eukaryotic genomes, presumably because ribosome of eukaryotes scans mRNA from the 5' to 3' direction to find proper start codons. Extra ATG triplets before start codons would confuse the process and thus they have been negatively selected in eukaryotic genomes. In eubacterial genomes, on the other hand, ribosome binds to the Shine-Dalgarno (SD) sequence at once without mRNA scanning, and the characteristic patterns of ATG triplet depletion were not observed (Saito, R., Tomita, M., 1999. On negative selection against ATG triplets near start codons in eukaryotic and procaryotic genomes. J. Mol. Evol. 48, 213-217). The ATG triplet analysis on archaebacterial genomes revealed that Methanococcus jannaschii and Pyrococcus horikoshii show patterns similar to eukaryotes, implying that these species employ scanning of mRNA from the 5' to 3' direction in the process of translation initiation. On the other hand, our earlier study found that these archaea have SD-like sequences, which are complementary to the 3' end sequence of 16S rRNA, as in eubacterial translation initiation (Osada, Y., Saito, R., Tomita, M. Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various procaryotes. Bioinformatics, in press). These two results combined lead us to conclude that these archaea probably use a hybrid mechanism; their ribosome scans mRNAs from the 5' to 3' direction and then 16S rRNA binds to the SD-like sequence of the 5' UTR.