A second eukaryotic group with mitochondrion-encoded tmRNA: in silico identification and experimental confirmation.

In bacteria, stalled ribosomes are rescued by transfer-mRNA (tmRNA) that catalyzes two steps. First, a non-encoded alanine is added to the incomplete polypeptide chain by the tRNA (Ala) -like portion of tmRNA, and second, the ribosome switches to the mRNA-like domain of tmRNA, thus resuming protein synthesis. Mitochondrial DNA (mtDNA)-encoded mt-tmRNA is so far only known from jakobid protists, but we posit that the corresponding ssrA gene may also reside in other mtDNAs. Here we present a highly sensitive covariance model built from jakobid ssrA genes that identifies previously unrecognized ssrA homologs in mtDNAs of oomycetes. These genes, located in previously unassigned genomic regions, are circular permuted as in α-Protobacteria, implying that pre-tmRNA is processed and the two pieces are held together by non-covalent interactions. RNA-Seq data from Phytophthora sojae confirm predicted processing sites as well as post-transcriptional addition of 3' CCA, a prerequisite for tmRNAs to be charged with alanine by alanyl-tRNA synthetase. Structure modeling of oomycete tmRNAs infers that the mRNA-like domain is lacking as in jakobids. Features of mitochondrial tmRNAs include the G-U pair at position three of the acceptor stem, a hallmark of bacterial tmRNAs, and a T-loop sequence that differs from that of standard tRNAs and most bacterial tmRNAs, forming alternative, virtually isosteric tertiary interactions with the D-loop. The anticodon stem has two additional G-A base pairs formed between the D-loop and the variable region, shortening the length of the variable region to a single nucleotide.

The long D-stem of the selenocysteine tRNA provides resilience at the expense of maximal function.

BACKGROUND: The selenocysteine tRNA (tRNASec) has a uniquely long D-stem containing 6 base pairs. RESULTS: The extended D-stem is not essential for function but is required for stability. CONCLUSION: Enhanced secondary structure in selenocysteine tRNA compensates for the absence of canonical tertiary interactions. SIGNIFICANCE: The flexibility due to the absence of tertiary interactions is required for tRNASec function, whereas the enhanced secondary structure compensates for the decreased stability. The D-stem of the selenocysteine tRNA (tRNA(Sec)) contains 2 additional base pairs, which replace tertiary interactions 8-14 and 15-48 universally present in all other cytosolic tRNAs. To study the role of these additional base pairs in the tRNA(Sec) function, we used the instant evolution approach. In vivo screening of six combinatorial gene libraries provided 158 functional variants of the Escherichia coli tRNA(Sec). Analysis of these variants showed that the additional base pairs in the D-stem were not required for the tRNA(Sec) function. Moreover, at lower temperatures, these base pairs notably harmed the tRNA(Sec) activity. However, at elevated temperatures, these base pairs became essential as they made the tRNA structure more stable. The alternative way to stabilize the structure through formation of the standard tertiary interactions was not an option for tRNA(Sec) variants, which suggests that the absence of these interactions and the resulting flexibility of the tertiary structure are essential for tRNA(Sec) function.

Twist-joints and double twist-joints in RNA structure.

Analysis of available RNA crystal structures has allowed us to identify a new family of RNA arrangements that we call double twist-joints, or DTJs. Each DTJ is composed of a double helix that contains two bulges incorporated into different strands and separated from each other by 2 or 3 bp. At each bulge, the double helix is over-twisted, while the unpaired nucleotides of both bulges form a complex network of stacking and hydrogen-bonding with nucleotides of helical regions. In total, we identified 14 DTJ cases, which can be combined in three groups based on common structural characteristics. One DTJ is found in a functional center of the ribosome, another DTJ mediates binding of the pre-tRNA to the RNase P, and two more DTJs form the sensing domains in the glycine riboswitch.

Recurrent RNA motifs as probes for studying RNA-protein interactions in the ribosome.

To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.

The adenosine wedge: a new structural motif in ribosomal RNA.

Here, we present a new recurrent RNA arrangement, the so-called adenosine wedge (A-wedge), which is found in three places of the ribosomal RNA in both ribosomal subunits. The arrangement has a hierarchical structure, consisting of elements previously described as recurrent motifs, namely, the along-groove packing motif, the A-minor and the hook-turn. Within the A-wedge, these elements are involved in different types of cause-effect relationships, providing together for the particular tertiary structure of the motif.

A hierarchical model for evolution of 23S ribosomal RNA.

The emergence of the ribosome constituted a pivotal step in the evolution of life. This event happened nearly four billion years ago, and any traces of early stages of ribosome evolution are generally thought to have completely eroded away. Surprisingly, a detailed analysis of the structure of the modern ribosome reveals a concerted and modular scheme of its early evolution.

Active suppressor tRNAs with a double helix between the D- and T-loops.

One of the most conserved elements of the tRNA structure is the reverse-Hoogsteen base-pair T54--A58 in the T-loop, which plays a major role in the maintenance of the standard L-shape conformation. Here, we present the results of in vivo selection of 51 active suppressor tRNA clones, none of which contains base-pair T54--A58. In 49 clones, we found two regions in the D and T-loops that are complementary to each other. This finding suggests the existence of an inter-loop double helix consisting of three base-pairs, which could have the same role as base-pair T54--A58 in the fixation of the juxtaposition of the two helical domains within the L-shape. From this point of view, the appearance of the inter-loop double helix represents a compensatory effect for the absence of base-pair T54--A58. The results shed new light on the role of different elements of the tRNA structure in the formation of the standard L-shape conformation and on the possibility of synonymous replacements of one arrangement by another in functional RNA molecules.

The three transfer RNAs occupying the A, P and E sites on the ribosome are involved in viral programmed -1 ribosomal frameshift.

The -1 programmed ribosomal frameshifts (PRF), which are used by many viruses, occur at a heptanucleotide slippery sequence and are currently thought to involve the tRNAs interacting with the ribosomal P- and A-site codons. We investigated here whether the tRNA occupying the ribosomal E site that precedes a slippery site influences -1 PRF. Using the human immunodeficiency virus type 1 (HIV-1) frameshift region, we found that mutating the E-site codon altered the -1 PRF efficiency. When the HIV-1 slippery sequence was replaced with other viral slippery sequences, mutating the E-site codon also altered the -1 PRF efficiency. Because HIV-1 -1 PRF can be recapitulated in bacteria, we used a bacterial ribosome system to select, by random mutagenesis, 16S ribosomal RNA (rRNA) mutations that modify the expression of a reporter requiring HIV-1 -1 PRF. Three mutants were isolated, which are located in helices 21 and 22 of 16S rRNA, a region involved in translocation and E-site tRNA binding. We propose a novel model where -1 PRF is triggered by an incomplete translocation and depends not only on the tRNAs interacting with the P- and A-site codons, but also on the tRNA occupying the E site.

G-ribo motif favors the formation of pseudoknots in ribosomal RNA.

Analysis of the pseudoknots existing in the ribosomal RNA showed that four of them are formed with the help of G-ribo, a recently identified RNA recurrent motif. The analysis of these pseudoknots revealed two major aspects in the G-ribo motif structure, which together provide the structural context favoring the formation of two different types of pseudoknots. The first aspect pertains to a particular side-by-side juxtaposition of two double helices that facilitates switches of the polynucleotide chain between different strands. The second aspect deals with the presence of an adenosine at a specific place where it can stabilize a particular arrangement of two quasicoaxial helices required for the pseudoknot formation. Additional analysis shows that the latter aspect is also present in other pseudoknots not related to the G-ribo motif or the ribosome, and thus represents a general structural element favoring the formation of pseudoknots.

Rational design of an estrogen receptor mutant with altered DNA-binding specificity.

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid-one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERalpha, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.

G-ribo: a new structural motif in ribosomal RNA.

Analysis of the available crystal structures of the ribosome and of its subunits has revealed a new RNA motif that we call G-ribo. The motif consists of two double helices positioned side-by-side and connected by an unpaired region. The juxtaposition of the two helices is kept by a complex system of tertiary interactions spread over several layers of stacked nucleotides. In the center of this arrangement, the ribose of a nucleotide from one helix is specifically packed with the ribose and the minor-groove edge of a guanosine from the other helix. In total, we found eight G-ribo motifs in both ribosomal subunits. The location of these motifs suggests that at least some of them play an important role in the formation of the ribosome structure and/or in its function.

Close packing of helices 3 and 12 of 16 S rRNA is required for the normal ribosome function.

The along-groove packing motif is a quasi-reciprocal arrangement of two RNA double helices in which a backbone of each helix is closely packed within the minor groove of the other helix. At the center of the inter-helix contact, a GU base pair in one helix packs against a Watson-Crick base pair in the other helix. Here, based on in vivo selection from a combinatorial gene library of 16 S rRNA and on functional characterization of the selected clones, we demonstrate that the normal ribosome performance requires that helices 3 and 12 be closely packed. In some clones the Watson-Crick and GU base pairs exchange in their positions between the two helices, which affects neither the quality of the helix packing, nor the ribosome function. On the other hand, perturbations in the close packing usually lead to a substantial drop in the ribosome activity. The functionality of the clones containing such perturbations may depend on the presence of particular elements in the vicinity of the area of contact between helices 3 and 12. Such cases do not exist in natural 16 S rRNA, and their selection enriches our knowledge of the constraints imposed on the structure of ribosomal RNA in functional ribosomes.

Specific and non-specific purine trap in the T-loop of normal and suppressor tRNAs.

To elucidate the general constraints imposed on the structure of the D and T-loops in functional tRNAs, active suppressor tRNAs were selected in vivo from a combinatorial tRNA gene library in which several nucleotide positions in these loops were randomized. Analysis of the nucleotide sequences of the selected clones demonstrates that most of them contain combination U54-A58 allowing the formation of the standard reverse-Hoogsteen base-pair 54-58 in the T-loop. With only one exception, all these clones fall into two groups, each characterized by a distinct sequence pattern. Analysis of these two groups has allowed us to suggest two different types of nucleotide arrangement in the DT region. The first type, the so-called specific purine trap, is found in 12 individual tRNA clones and represents a generalized version of the standard D-T loop interaction. It consists of purine 18 sandwiched between the reverse-Hoogsteen base-pair U54-A58 and purine 57. The identity of purine 18 is restricted by the specific base-pairing with nucleotide 55. Depending on whether nucleotide 55 is U or G, purine 18 should be, respectively, G or A. The second structural type, the so-called non-specific purine trap, corresponds to the nucleotide sequence pattern found in 16 individual tRNA clones and is described here for the first time. It consists of purine 18 sandwiched between two reverse-Hoogsteen base-pairs U54-A58 and A55-C57 and, unlike the specific purine trap, requires the T-loop to contain an extra eighth nucleotide. Since purine 18 does not form a base-pair in the non-specific purine trap, both purines, G18 and A18, fit to the structure equally well. The important role of both the specific and non-specific purine traps in the formation of the tRNA L-shape is discussed.

Key elements in maintenance of the tRNA L-shape.

Based on in vivo selection of effective suppressor tRNAs from two different combinatorial gene libraries in which several nucleotides in the D and T-loops were randomized, we show that the position of the reverse-Hoogsteen base-pair in the T-loop, normally formed between nucleotides 54-58, co-varies with the length of the D-domain. When the D-domain has the normal length, the position of the reverse-Hoogsteen base-pair in the T-loop is always such that it allocates two unpaired nucleotides 59-60 for the bulge that fills the space between the D and T-domains. However, when the D-domain becomes shorter, the position of the reverse-Hoogsteen base-pair changes in the way that more nucleotides are now allocated to the T-loop bulge, so that the total length of the D-domain and of the bulge remains the same. Such compensation guarantees that in all tRNAs, the D and T-domains are always juxtaposed in the standard way. It also demonstrates the major role of the two T-loop elements, the bulge and the reverse-Hoogsteen base-pair, in the formation of the canonical tRNA L-shape.

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.

The frameshift stimulatory signal of human immunodeficiency virus type 1 group O is a pseudoknot.

Human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshift to produce Gag-Pol, the precursor of its enzymatic activities. This frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2nt (loop 1) and 20nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the frameshift efficiency to near wild-type level. The difference between the frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission.

Importance of the reverse Hoogsteen base pair 54-58 for tRNA function.

To elucidate the general constraints imposed on the structure of the D- and T-loops in functional tRNAs, active suppressor tRNAs were selected in vivo from a combinatorial tRNA gene library in which several nucleotide positions of these loops were randomized. Analysis of the nucleotide sequences of the selected clones demonstrates that among the randomized nucleotides, the most conservative are nucleotides 54 and 58 in the T-loop. In most cases, they make the combination U54-A58, which allows the formation of the normal reverse Hoogsteen base pair. Surprisingly, other clones have either the combination G54-A58 or G54-G58. However, molecular modeling shows that these purine-purine base pairs can very closely mimic the reverse Hoogsteen base pair U-A and thus can replace it in the T-loop of a functional tRNA. This places the reverse Hoogsteen base pair 54-58 as one of the most important structural aspects of tRNA functionality. We suggest that the major role of this base pair is to preserve the conformation of dinucleotide 59-60 and, through this, to maintain the general architecture of the tRNA L-form.

GU receptors of double helices mediate tRNA movement in the ribosome.

A new RNA structural motif consisting of two double helices closely packed via minor grooves is found in many places in the ribosome structure. The packing requires that a GU base pair in one helix be packed against a Watson-Crick pair in the other helix. Two such motifs mediate the interaction of the P- and E-tRNA with the large ribosomal subunit. Analysis of the particular positions of these two motifs in view of the available data on occupancy of tRNA-binding sites and structural changes in the ribosome during the elongation cycle suggests a distinct role for each motif in tRNA translocation.