Hyouta Himeno

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post‐transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass‐silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5‐methyluridine and pseudouridine. Both modifications are located within the proposed tRNA‐like domain, in a seven‐nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem–loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post‐transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Strand-specific nucleotide composition bias in echinoderm and vertebrate mitochondrial genomes.

SummaryThe gene organization of starfish mitochondrial DNA is identical with that of the sea urchin counterpart except for a reported inversion of an approximately 4.6-kb segment containing two structural genes for NADH dehydrogenase subunits 1 and 2 (ND 1 and ND 2). When the codon usage of each structural gene in starfish, sea urchin, and vertebrate mitochondrial DNAs is examined, it is striking that codons ending in T and G are preferentially used more for heavy strand-encoded genes, including starfish ND 1 and ND 2, than for light strand-encoded genes, including sea urchin ND 1 and ND 2. On the contrary, codons ending in A and Care preferentially used for the light strand-encoded genes rather than for the heavy strand-encoded ones. Moreover, G-U base pairs are more frequently found in the possible secondary structures of heavy strandencoded tRNAs than in those of light strand-encoded tRNAs. These observations suggest the existence of a certain constraint operating on mitochondrial genomes from various animal phyla, which results in the accumulation of G and T on one strand, and A and C on the other.

Requirement of transfer‐messenger RNA for the growth of Bacillus subtilis under stresses

Bacterial transfer‐messenger RNA (tmRNA, 10Sa RNA) is involved in a trans‐translation reaction which contributes to the degradation of incompletely synthesized peptides and to the recycling of stalled ribosomes. However, its physiological role in the cell remains elusive. In this study, an efficient system for controlling the expression of the gene for tmRNA (ssrA), as well as a tmRNA gene‐defective strain (ssrA::cat), were constructed in Bacillus subtilis. The effects of tmRNA on the growth of the cells were investigated under various physiological culture conditions using these strains.

Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single‐stranded RNAs

A novel translation, trans‐translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans‐translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional.

The Role of anticodon bases and the discriminator nucleotide in the recognition of some E. coli tRNAs by their aminoacyl-tRNA synthetases

SummaryThe T7 polymerase transcription system was used for in vitro synthesis of unmodified versions of the E. coli tRNA mutants that insert asparagine, cysteine, glycine, histidine, and serine. These tRNAs were used to qualitatively explore the role of some anticodon bases and the discriminator nucleotide in the recognition of tRNA by aminoacyl-tRNA synthetases. Coupled with data from earlier studies, these new results essentially complete a survey of all E. coli tRNAs with respect to the involvement of anticodon bases and the discriminator nucleotide in tRNA recognition. It is found that in the vast majority of tRNAs both of these elements are significant components of tRNA identity. This is not universally true, however. Anticodon sequences are unimportant in tRNAser, tRNALeu, and tRNAAla while the discriminator base is inconsequential in tRNAser and tRNAThr. The significance of these results for origin-of-life studies is discussed.

Determinants on tmRNA for initiating efficient and precise trans-translation: some mutations upstream of the tag-encoding sequence of Escherichia coli tmRNA shift the initiation point of trans-translation in vitro.

tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNAs tag sequence. The mechanism underlying resumption of translation at a definite position is not known. In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis. One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation. Any 1-nt deletion within the span of -5 to -1, but not at -6, made the frame of tag translation heterologous. Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of -4 to -2. In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities. A possible stem and loop structure around this region was not required for transtranslation. It was concluded that the tag translation requires the primary sequence encompassing -6 to +11, in which the central 3 nt, A-4, G-3, and U-2, play an essential role. It was also found that several base substitutions within the span of -6 to -1 extensively shifted the tag-initiation point by -1.

Interaction of SmpB with ribosome from directed hydroxyl radical probing

To add a tag-peptide for degradation to the nascent polypeptide in a stalled ribosome, an unusual translation called trans-translation is facilitated by transfer-messenger RNA (tmRNA) having an upper half of the tRNA structure and the sequence encoding the tag-peptide except the first alanine. During this event, tmRNA enters the vacant A-site of the stalled ribosome without a codon–anticodon interaction, but with a protein factor SmpB. Here, we studied the sites and modes of binding of SmpB to the ribosome by directed hydroxyl radical probing from Fe(II) tethered to SmpB variants. It revealed two SmpB-binding sites, A-site and P-site, on the ribosome. Each SmpB can be superimposed on the lower half of tRNA behaving in translation. The sites of cleavages from Fe(II) tethered to the C-terminal residues of A-site SmpB are aligned along the mRNA path towards the downstream tunnel, while those of P-site SmpB are found almost exclusively around the region of the codon–anticodon interaction in the P-site. We propose a new model of trans-translation in that the C-terminal tail of SmpB initially recognizes the decoding region and the mRNA path free of mRNA by mimicking mRNA.

Identity determinants of E. coli threonine tRNA

To investigate the identity determinants of E. coli threonine tRNA, various transcripts were prepared by in vitro transcription system with T7 RNA polymerase. Substitutions of the anticodon second letter G35 and the third letter U36 to other nucleotides led to a remarkable decrease of threonine charging activity. Charging experiments with a series of anticodon-deletion transcripts also suggest the importance of the G35U36 sequence. A mutation at either the G1-C72 or C2-G71 base pair in the acceptor stem seriously affected the threonine charging activity. These results indicate that the second and third positions of the anticodon and the first and second base pairs in the acceptor stem are the recognition sites of E. coli tRNA(THR) for threonyl-tRNA synthetase. Discriminator base, A73, is not involved in threonine charging activity.

Detection of tmRNA-mediated trans-translation products in Bacillus subtilis.

Bacterial tmRNA (10Sa RNA) is involved in a trans‐translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans‐translation.

Interaction of 10Sa RNA with ribosomes in Escherichia coli

10Sa RNA is a bacterial small stable RNA, in which the 5′‐ and 3′‐end sequences are folded into a tRNA‐like structure. The RNA accepts alanine in vitro, and interacts with 70S ribosomes in the cells. In this study, we examined the ribosome‐binding properties of Escherichia coli 10Sa RNA in vivo, and found that the aminoacylation ability of 10Sa RNA with alanine is necessary for the binding to 70S ribosomes. 10Sa RNA was also found to bind only to 70S monosomes and not to polysomes. Recently, E. coli 10Sa RNA was suggested to be used as mRNA for tag peptides, which were found to attach to the C‐termini of truncated peptides synthesized in vivo. The present results are consistent with the ‘trans‐translation’ model, which has been proposed for tag‐peptide synthesis.