A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli.

Abstract

We have determined that lOSa RNA (one of the small stable RNAs found in Escherichia cobl) has an interesting structural feature: the 5' end and the 3' end of lOSa RNA can be arranged in a s ture that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of lOSa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to lOSa RNA (pre-lOSa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding lOSa RNA (ssrA) caused a reduction in the rate of cell growth, which was especaly apparent at 45C, and a reduction in motility on se d agar. These phenotypic characteristics of the deletion strain (AssrA) allowed us to investigate the effects of some mutations in lOSa RNA in vivo, although the exact function of lOSa RNA still remains unclear. When the G-U pair (G3-U357) in lOSa RNA, which may be equivalent to the determinant G-U pair of alanine tRNA, was changed to a GA or G-C pair, the ability to complement the phenotypic mutations of the AssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a GA pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (aaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of lOSa RNA are indeed manifested in vivo.lOSa RNA is one of the small stable RNAs that is found in Escherichia coli. It was first identified as lOS RNA, which was actually a mixture of lOSa RNA and Ml RNA (originally designated lOSb RNA) (1, 2). Subsequently, Ml RNA was shown to be a "catalytic RNA" (3), and it has been extensively studied from both a structural and a functional perspective (4). However, in contrast to Ml RNA, lOSa RNA has not been the focus of much recent attention, and very little is known about either its structure or its function.In our previous study, we found that the ssrA gene that encodes lOSa RNA of E. coli is included in the genomes of phages 438, 439, and 440 of Kohara's library (5), which corresponds to 56.5 minutes on the linkage map of the chromosome (6). We cloned a 2.2-kb fragment that covers the overlapping region of these phages into a plasmid vector and determined the nucleotide sequence of the fragment. § We also purified lOSa RNA from E. coli strain W3110 and determined its nucleotide sequence from both the 5' and the 3' end.The nucleotide sequence of the ssrA gene from E. coli has already been reported by Chauhan and Apirion (7). In comparing our sequences with the published sequence, we noticed an interesting structural feature of lOSa RNA, as presented in Fig. 2-namely, 7 nucleotides at the 5' end and 28 nucleotides at the 3' end can be arranged in a structure that is equivalent to a half-molecule of tRNA. This half-mole...

Identification of Endogenous SsrA-tagged Proteins Reveals Tagging at Positions Corresponding to Stop Codons

“…Deletions of SsrA or SmpB are lethal in some bacteria and cause slow growth under stress conditions in other bacteria (10 -12). In E. coli, for instance, the growth defects of strains lacking SsrA are magnified at high temperatures or under carbon starvation (2,13), suggesting that quality assurance becomes more important when the incidence of translational errors is elevated. Other phenotypes of SsrA-defective E. coli include reduced motility (2), increased protease activity (14), defects in plating of phage immP22 and induction of temperature-sensitive lysogens of phage Mu (6,15), and increased levels of LacI, cI, and LexA repression (16).…”

Protein factors associated with the SsrA⋅SmpB tagging and ribosome rescue complex

“…SsrA RNA is charged with alanine by alanyl-tRNA synthetase (1), and aminoacylated SsrA forms a ternary complex with elongation factor (EF)-Tu and GTP (8,9). Complex formation with EFTu⅐GTP protects the labile ester linkage of charged SsrA (9) and, by analogy with charged tRNAs, undoubtedly is important for delivery of alanyl-SsrA to ribosomes.…”

“…tmRNA ͉ 10Sa RNA ͉ trans translation ͉ protein-RNA recognition ͉ VacB I n bacteria, SsrA RNA (also known as tmRNA or 10Sa RNA) acts both as a tRNA and an mRNA in a process that clears stalled ribosomes and tags the nascent polypeptides associated with such ribosomes with a C-terminal peptide that results in their degradation (1)(2)(3). The 5Ј and 3Ј ends of SsrA RNA form an alanyl-tRNA-like domain with a disrupted stem replacing the normal tRNA anticodon stem-loop (4,5).…”

Lost in translation

“…Parfois même, la bactérie est incapable de survivre lorsque la trans-traduction est affectée, lors d'infections comme la blennorragie par exemple (agent responsable : N. gonorrhae), tandis que d'autres espèces tolèrent l'absence de trans-traduction (E. coli ou B. subtilis par exemple) [23][24][25][26][27]. Bien que la trans-traduction ne soit pas systématiquement indispensable à la survie bactérienne, son absence, par inactivation génétique du couple ARNtm-SmpB, rend cependant les cellules plus sensibles à la présence de certains antibiotiques, même à des concentrations sublétales [28].…”