The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination.

The deletion of the highly conserved cytidine nucleotide at position 1054 in E. coli 16S rRNA has been characterized to confer an UGA stop codon specific suppression activity which suggested a functional participation of small subunit rRNA in translational termination. Based on this structure-function correlation we constructed the three point mutations at site 1054, changing the wild-type C residue to an A, G or U base. The mutations were expressed from a complete plasmid encoded rRNA operon (rrnB) using a conditional expression system with the lambda PL-promoter. All three altered 16S rRNA molecules were expressed and incorporated into 70S ribosomal particles. Structural analysis of the protein and 16S rRNA moieties of the mutant ribosomes showed no differences when compared to wild-type particles. The phenotypic analysis revealed that only the 1054G base change led to a significantly reduced generation time of transformed cells, which could be correlated with the inability of the mutant ribosomes to specifically stop at UGA stop codons in vivo. The response towards UAA and UAG termination codons was not altered. Furthermore, in vitro RF-2 termination factor binding experiments indicated that the association behaviour of mutant ribosomes was not changed, enforcing the view that the UGA stop codon suppression is a direct consequence of the rRNA mutation. Taken together, these results argue for a direct participation of that 16S rRNA motif in UGA dependent translational termination and furthermore, suggest that termination factor binding and stop codon recognition are two separate steps of the termination event.     

A rRNA-mRNA base pairing model for UGA-dependent termination.

A series of site-directed mutations has been constructed in E coli 16S rRNA and shown to suppress UGA-dependent translational termination. With the exception of the C726 to G base change, all were constructed in helix 34. Characterization of these mutations is reviewed here and from these data and mRNA-rRNA base pairing model for the termination event is presented. The interaction functions via antiparallel base pairing between either 1 of the 2 UCA motifs in helix 34 and the complementary UGA stop codon on the message, thus forming a quasicontinuous A-type helical structure that is further stabilized by stacking enthalpy. Finally, rRNA motifs potentially required for UAA and UAG-dependent translational termination are discussed.     

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.     

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.     

Translational fidelity mutations in 18S rRNA affect the catalytic activity of ribosomes and the oxidative balance of yeast cells

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.     

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.     

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.     

Ribosomal RNA and peptidyl-tRNA hydrolase: a peptide chain termination model for lambda bar RNA inhibition.

We propose here a model to explain the inhibition of bacteriophage lambda (lambda) vegetative growth and the killing of E coli cells defective in peptidyl-tRNA hydrolase (Pth) by lambda bar RNA. The model suggests that bar RNA, which contains a characteristic UGA triplet, base-pairs in an anti-parallel fashion with the 1199-1205 region of E coli 16S rRNA. In doing so, it prevents the required functioning of that region of 16S rRNA in UGA-specific peptide chain termination. Pth is implicated in peptide chain termination because a defect in Pth is required for the achievement of the bar RNA inhibitory effects. We make certain predictions that flow from the model, predictions involving suppression of nonsense mutations, and present preliminary experimental results that demonstrate the fulfillment of those predictions.     

Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.     

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.     

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.     

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.     

An rRNA Fragment and Its Antisense Can Alter Decoding of Genetic Information

rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in E. coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.     

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.     

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.     

On the role of the termination factor RF-2 and the 16S RNA in protein synthesis

In the translational termination step of protein synthesis the three termination codons UAA, UAG or UGA are recognized by so-called release or termination factors. The release factor RF-1 interacts with UAG and UAA whereas RF-2 is specific for UGA and UAA. Two mechanisms concerning the termination event have been discussed so far: recognition of the termination codon by the protein in a tRNA-like manner or double-strand formation between the codon and the 3' end of the 16S rRNA which is stabilized by the termination factor. Using equilibrium dialysis we show that 40% of the ribosomes can bind UGAA in an RF-2-dependent manner. The stability with the correct combination RF-2-UGA is tenfold higher as compared to the wrong termination codon UAG. We confirm prior findings that the termination factor RF-2 is bound to the A-site of the ribosome. In addition to the ribosomal proteins L2, L10, L7/L12 and L20 of the large subunit and S6 and S18 of the small subunit, the 16S rRNA became labelled when radioactive UGA was crosslinked to the ribosome in the presence of RF-2. Our data support a mechanism of termination in which a double strand between the termination codon and the 3' end of the 16S rRNA is formed as the starting event. The resulting RNA-RNA double strand in turn may be recognized and stabilized by the termination factor.