Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5'-leader

The tobacco etch virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.     

The influence of base identity and base pairing on the function of the alpha-sarcin loop of 23S rRNA.

The alpha-sarcin loop of large subunit rRNAs is one of the sites of interaction of elongation factors with the ribosome, and the target of the cytotoxins alpha-sarcin and ricin. Using a genetic selection for increased frameshifting in a reporter gene, we have isolated a C --> U mutation at position 2666 in the alpha-sarcin loop. In the NMR-derived structure of the loop, bases equivalent to 2666 and 2654 are paired via a non-canonical base pairing interaction. Each of the three base substitutions at C2666 and A2654 was constructed by site-directed mutagenesis of a plasmid borne copy of the rrnB operon of Escherichia coli. Only the C2666 --> U and A2654 --> G mutations that resulted in the formation of canonical A-U and C-G base pairs respectively, increased the levels of stop codon readthrough and frameshifting. The effects of different base pair combinations at positions 2666 and 2654 on ribosome function were then tested by constructing and analyzing all possible base combinations at these sites. All A --> G base substitution mutations at position 2654 and C --> U substitutions at position 2666 increased the levels of translational errors. However, these effects were greatest when G2654 and U2666 had the potential to engage in standard Watson-Crick base pairing interactions. These data indicate that base identity as well as base pairing interactions are important for the function of this essential component of the large subunit rRNA.     

Potential new antibiotic sites in the ribosome revealed by deleterious mutations in RNA of the large ribosomal subunit

The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.     

How to avoid undesirable interactions during ribosome assembly?

Ribosome subunit assembly in bacteria is assisted by several non‐ribosomal proteins, the absence of which leads to assembly defects. The two DEAD‐box RNA helicases SrmB and DeaD/CsdA are required for efficient assembly of the ribosome large subunit, in particular at low temperature, but their sites of action on rRNA were not known until now. In this issue of Molecular Microbiology, Proux et al. show that SrmB acts far away from its tethering site on the assembly intermediate particle. A genetic screen identified mutations in complementary sequences of 23S and 5S rRNA that help to bypass SrmB deficiency, partially correcting the large subunit assembly defect. The results suggest that 5S rRNA and 23S rRNA can interact via base‐pairing, forming a non‐native structure that needs to be corrected. The authors discuss attractive hypotheses on SrmB acts during large subunit assembly.     

Occurrence and spacing of ribosome recognition sites in mRNAs of chloroplasts from higher plants

A computer-aided search for potential ribosome recognition sequences of mRNAs from tobacco chloroplasts shows that more than 90% of mRNA species contain sequences upstream of the respective initiator codons, which allow base pairing with 3′-terminal sequences of small subunit rRNA. This complementarity in several cases involves 16 S rRNA sequences between the canonical CCUCC sequence and the 3′-terminal stem/loop structure. The distances between potential ribosome recognition sequences and initiator codons can be up to 25 nucleotides which is much greater when compared to the spacing of 7±2 nucleotides observed for the classical Shine-Dalgarno sequences in bacterial mRNAs.     

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.     

Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning.

Helix 2 of the central pseudoknot structure in Escherichia coli 16S rRNA is formed by a long-distance interaction between nt 17-19 and 918-916, resulting in three base pairs: U17-A918, C18-G917and A19-U916. Previous work has shown that disruption of the central base pair abolishes ribosomal activity. We have mutated the first and last base pairs and tested the mutants for their translational activity in vivo , using a specialized ribosome system. Mutations that disrupt Watson-Crick base pairing result in strongly impaired translational activity. An exception is the mutation U916-->G, creating an A.G pair, which shows almost no decrease in activity. Mutations that maintain base complementarity have little or no impact on translational efficiency. Some of the introduced base pair substitutions substantially alter the stability of helix 2, but this does not influence ribosome functioning, neither at 42 nor at 28 degrees C. Therefore, our results do not support models in which the pseudoknot is periodically disrupted. Rather, the central pseudoknot structure is suggested to function as a permanent structural element necessary for proper organization in the center of the 30S subunit.     

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.     

The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin

We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila. The gene encodes an RNA molecule which is 1753 nucleotides in length. The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs. We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin. In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure. Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.     

Study of the functional interaction of the 900 Tetraloop of 16S ribosomal RNA with helix 24 within the bacterial ribosome

The 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) is amongst the most conserved regions of rRNA. This tetraloop forms a GNRA motif that docks into the minor groove of three base-pairs at the bottom of helix 24 of 16S rRNA in the 30S subunit. Both the tetraloop and its receptor in helix 24 contact the 23S rRNA, forming the intersubunit bridge B2c. Here, we investigated the interaction between the 900 tetraloop and its receptor by genetic complementation. We used a specialized ribosome system in combination with an in vivo instant evolution approach to select mutations in helix 24 compensating for a mutation in the 900 tetraloop (A900G) that severely decreases ribosomal activity, impairing subunit association and translational fidelity. We selected two mutants where the G769-C810 base-pair of helix 24 was substituted with either U-A or C x A. When these mutations in helix 24 were investigated in the context of a wild-type 900 tetraloop, the C x A but not the U-A mutation severely impaired ribosome activity, interfering with subunit association and decreasing translational fidelity. In the presence of the A900G mutation, both mutations in helix 24 increased the ribosome activity to the same extent. Subunit association and translational fidelity were increased to the same level. Computer modeling was used to analyze the effect of the mutations in helix 24 on the interaction between the tetraloop and its receptor. This study demonstrates the functional importance of the interaction between the 900 tetraloop and helix 24.     

Functional studies of the 900 tetraloop capping helix 27 of 16S ribosomal RNA

The 900 tetraloop (positions 898-901) of Escherichia coli 16S rRNA caps helix 27, which is involved in a conformational switch crucial for the decoding function of the ribosome. This tetraloop forms a GNRA motif involved in intramolecular RNA-RNA interactions with its receptor in helix 24 of 16S rRNA. It is involved also in an intersubunit bridge, via an interaction with helix 67 in domain IV of 23S rRNA. Using a specialized ribosome system and an instant-evolution procedure, the four nucleotides of this loop were randomized and 15 functional mutants were selected in vivo. Positions 899 and 900, responsible for most of the tetraloop/receptor interactions, were found to be the most critical for ribosome activity. Functional studies showed that mutations in the 900 tetraloop impair subunit association and decrease translational fidelity. Computer modeling of the mutations allows correlation of the effect of mutations with perturbations of the tetraloop/receptor interactions.     

Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome

Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.     

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 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.     

Interaction between the ribosomal subunits: 16S rRNA suppressors of the lethal ΔA1916 mutation in the 23S rRNA of <Emphasis Type="Italic">Escherichia coli</Emphasis>

A1916 in 23S rRNA is located in one of the major intersubunit bridges of the 70S ribosome. Deletion of A1916 disrupts the intersubunit bridge B2a, promotes misreading of the genetic code and is lethal. In a genetic selection for suppressor mutations, two base substitutions in 16S rRNA were recovered that restored viability and also allowed expression of ΔA1916-associated capreomycin resistance. These mutations were G1048A in helix 34 and U1471C in helix 44. Restoration of function is incomplete, however, and the double mutants are slow-growing, defective in subunit association and support high levels of translational errors. In contrast, none of these parameters is affected by the single 16S suppressor mutations. U1471C likely affects another intersubunit contact, bridge B6, suggesting that interactions between different bridges and cross-talk between subunits contributes to ribosomal function.     

Selection for intragenic suppressors of lethal 23S rRNA mutations in Escherichia coli identifies residues important for ribosome assembly and function

Mutations in several functionally important regions of the 23S rRNA of E. coli increase the levels of frameshifting and readthrough of stop codons. These mutations include U2555A, U2555G, ΔA1916 and U2493C. The mutant rRNAs are lethal when expressed at high levels from a plasmid, in strains also expressing wild type rRNA from chromosomal rrn operons. The lethal phenotype can be suppressed by a range of second-site mutations in 23S rRNA. However, analysis of the functionality of the double mutant rRNAs in heterogeneous ribosome populations shows that in general, the second site mutations do not restore function. Instead, they prevent the assembly, or entry of the mutant 50S subunits into the functioning 70S ribosome and polysome pools, by affecting the competitiveness of the mutant subunits for association with 30S particles. The second-site mutations lie in regions of the 23S rRNA involved in subunit assembly, intersubunit bridge formation and interactions of the ribosome with tRNAs and factors. These second site suppressor mutations thus define functionally important rRNA nucleotides and this approach may be of general use in the functional mapping of large RNAs.     

Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA

Previously, we characterized two host protein binding elements located within the 3'-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3'-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).     

Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction

Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA. As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance. A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool. A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA. In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations. These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome. This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop.     

A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit

A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40-50% between 30 degrees C and 42 degrees C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 A deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein-RNA interactions elsewhere in the ribosome.