JMB J. Mol. Biol. (1997) 268, 803-808 _@ COMMUNICATION In Vitro Trans Translation Mediated by Alanine- charged 10Sa RNA Hyouta Himeno, Masakazu Sato, Toshimasa Tadaki, Masaaki Fukushima, Chisato Ushida and Akira Muto* Department of Biology, Faculty of Science, Hiroseki University Hirosaki 036, Japan *Comespoming author 10Sa RNA is a bacterial small stable RNA, in which the 5’ and 3-terminal sequences can be folded into a (RNAJike secondary structure which can be aminoacylated with alanine. It was found that Escherichia colt 10Sa RNA facilitated the incorporation of alanine, tyrosine, aspartic acid and glutamic acid, but not valine, isoleucine, serine or arginine, into the growing polypeptide in_vitro, depending on poly (Uj-directedpoly- phenylalanine synthesis. This result indicates that 10Sa RNA functions as an mRNA for the tag-peptide which has been found to be attached to the C termini of truncated polypeptides synthesized in veo. Aminoacylation with alanine was required for tag-specific amino acid incorporation and for efficient association of 105a RNA with the ribosome, indicating that 10Sa RNA also functions as an alanine tRNA in the tag-peptide synthesis. The dual function of 108a RNA both as an mRNA and as a {RNA in vilro strongly supports the trans translation hypothesis © 1997 Academic Press Limited Keywords: 108a RNA; trans translation; tmRNA; tag-peptide; translation 10Sa RNA is a small stable bacterial RNA found in Escherichia coli and many other bacteria (Ray & Apirion, 1979; Lee et al, 1979; Brown et al., 1990; ‘Tyagi & Kinger, 1991; Ushida et al., 1994; Komine ef al, 1994; Haring ef al., 1995; Brosius, 1996). The 5 and terminal sequences in all 10Sa RNAs can be folded into a tRNA-like secondary structure comprising the 3-terminal CCA, a TWC arm and an amino acid acceptor stem with a G-U wobble base-pair at the third base pair position, which is known as an alanine acceptor identity determinant (Figure 1; Ushida ef al., 1994; Komine et al, 1994), Indeed, E. coli and Bacillus subtilis 10Sa RNAs can be aminoacylated with alanine in vitro (Ushida et al., 1994; Komine ef al., 1994), and interact predo- minanily with 70$ ribosome in vio (Ushida of al. 1994; Komine et al., 1996; Tadaki et al,, 1996). This RNA is therefore presumed to play some role in protein synthesis. Recently, F. coli 10Sa RNA has been shown to in- clude a small open reading frame (Figure 1), the last ten amino acid sequence of which is identical with that of the 11 amino acid tag-peptide (AlaAla- AsnAspGluAsnTyrAlaLeuAlaAla) found in the C fermini of truncated murine interleukin ex- pressed in E. coli (Tu et al,, 1995), An identical tag- peptide has also been found in the C termini of lambda cl repressor and cytochrome b-562 when (0022-2856 /97 /2008053-06 $25.00 /0/mb97 1011 they are translated from mRNAs lacking 4 termin- ation codon (Keiler et al., 1996). The tag-peptide ad- dition depends on the presence of the sivA gene that encodes 10Sa RNA (Tu ef al,, 1995; Keiler ef al., 1996), raising the possibility that 10Sa RNA itself acts as an mRNA for the tag-peptide. On the basis of these observations, Keiler et al. (1996) proposed a model for the mechanism of tag-pep- tide synthesis (fms translation). In this model, alanine-charged 10Sa RNA enters the tibosome when translation stops at the 3-end of the trun- cated mRNA lacking a stop codon. The 10Sa RNA donates its alanine to the stalled peptide chain, and then the tag is added to the C termi- nus of the nascent polypeptide chain by cotrans- lational switching of the ribosome from_ the damaged mRNA to the intemal sequence of 10Sa RNA. As a result, a chimeric protein of the trun- cated peptide and the tag-peptide connected by the alanine aminoacylated to 10Sa RNA is re- eased from the ribosome using the in-frame stop codon, allowing the ribosome to recycle. ‘The C-terminal tag-sequence is a signal for degradation by cellular proteases (Keiler et ai., 1996). A’ series of these reactions is thus thought to be a degradation system of a stalled polypeptide synthesis translated from a damaged mRNA (see also Jentsch, 1996; Atkins & Gesteland, 1996). (© 1987 Academie Press Limited804 10Sa RNA-dependent Trans Translation In Vitro MPRGGWPRKKPQK VANDENYALAA'* a Figure 1. A {RNA-like secondary structure and an open reading frame in F. coli 10Sa RNA. The length of this RNA is 363 nucleotides (Komine ef ai., 1994). The 5’ and 3-terminal sequences can be folded into a (RNA-like structure com- prising the amino acid acceptor stem, the TC arm and the 3-terminal CCA (Ushida ef al, 1994; Komine et al, 1994). An allemative secondary structure model based upon chemical and enzymatic probings was recently proposed (Felden et al, 1996a,b, 1997). A point mutation, designated by the arrow was made at the G-U base-pair in the amino acid acceptor stem, which is a major identity determinant of tRNA (McClain & Foss, 1988; Hou & Schimmel, 1988). An open reading frame shown by single-letter amino acid designation contains the last ten amino acids of the tag-peptide underlined, while the origin of the first alanine residue designated by parentheses has been unassigned (Tu et al, 1995). The re shown by asterisks. The region from nt 126 to nt 318 is represented as a termination eodans continuous line. The G-U or U-G wobble base-pairs are shown by small circles Here, we show that 10Sa RNA actually functions both as an alanine tRNA and an mRNA in vitro, which strongly supports the frms translation hypothesis. We examined the effect of 10Sa RNA on the in- corporation of alanine, a constituent of the tag-pep- tide, in an in vitro’ poly (U)-dependent poly- phenylalanine synthesis system using the $30 frac- tion extracted from an E, coli AssrA strain (Komine eh al., 1994). As shown in Figure 2(a), "C-labelled alanine was incorporated in the acid-insoluble frac- tion when 10Sa RNA was added to. the reaction mixture. This activity required poly (U) as an ex- ogenous mRNA, showing that poly-phenylalanine synthesis is necessary for 10Sa RNA-dependent alanine incorporation. Other tag-specific amino acids, tyrosine, aspartic acid and glutamic acid, were also incorporated in the presence of poly (U) (Figure 2(b) to 2(d)). In contrast, valine, whose codon GUC is present preceding the first alanine codon of the tag-sequence, was not incorporated (Figure 2(@)). Similarly, other tag-unrelated amino acids, isoleuicine, arginine and serine, were not in- corporated (Figure 2() to 2(h)). The absence of in- corporation of arginine, isoleucine or valine shows that the full-length translation of the potential open reading frame, which contains two arginine co- dons, one isoleucine codon and one valine codon (Figure 1), does not occur. The molar ratios of tyro- sine, aspartic acid or glutamic acid incorporated to alanine were about 0.2 to 0.25, which is consistent with the amino acid composition of the tag-pep- tide. These results strongly suggest that 10Sa RNA itself functions as an mRNA for tag-peptide syn- thesis, which is cotranslationally attached to the terminal end of poly-phenylalanine according to the frais translation model. Note that the poly (U) employed here was an mRNA that was free of a termination codon. The absence of incorporation of arginine, isoleucine and valine excludes the possibility that the tag-peptide synthesis involves a post-translational protein splicing (Shub — & Goodrich-Blair, 1992) between poly-phenylalanine and the product of the open reading, frame10Sa RIVA-dependent Trans Translation In Vitro 805 (a) (b) 40 8 4 6 a ee > f+, () (a) incorporated (pmol) (9) (h) 0 or 02 (O a1” 02 0 o1 02 0 or” 02 10Sa RNA (11g) Figure 2. 10Sa RNA-dependent amino acid incorporation i vitro, 10Sa RNA-dependent alanine (a), tyrosine (b), aspartic acid (0), glutamic acid (d), valine (e), isoleucine (0), arginine (g) and serine (h) incorporations in the presence (open circles) or absence (filled circles) of poly (U}. The £, coli 10Sa RNA gene ligated uncer the T7 RNA polymerase promoter sequence of the plasmid pGEMEX-2 was transformed into E. cali JM10® (DE3) which possesses the T7 RNA polymerase gene under the /ac promoter. 10a RNA, induced by the addition of 1.0 mM isopropylf3-thiogalactopyr- noside was purified as described (Ushida et al,, 1994). This averproduction system yielded an approximately 100- her amount of properly processed 108a RNA which was normally aminoacylated with alanine i vitro. The uubated $30 fraction was prepared from middle log phase cells of FE coli W3110 (AssrA) strain (Komine et a, 1994), as described (Oba et al, 1991). A typical reaction mixture (50 ul) contained $0 mM ‘Tris-HCl (pH 7.8), 5mM magnesium acetate, 150 mM ammonium chloride, 2.5 mM dithiothreitol, 1 mM ATP, 0.2 mM GTP, 5 mM phosphoe- nol pyruvate, 254g pyruvate kinase, 0.02mM ['Clalanine (62 GBq/mmol), ['Cltyrosine (167 GBq/mmol), ["Claspartic acid (7.88 GBq/mmel), (C}glutamic acid (10 GBq/mmol), ["CIvaline (9.62 GBq/mmol), ['4Clisoleucine (12 GBq/mmol}, ["Chserine (5.59 GBq/mmol) or ["*Clarginine (11.2 GBq/mmol), 0.05 mM each of the remaining un- Tabelied’ 19 amino acids, 0 to 012 yg of 10Sa RNA, and 10 lof the $30 fraction, in the presence ar absence of 25 jg of poly (U) Gigma, 50 to 100-mer). All of the labelled amino acids were purchased from Amersham. The reaction mix- ture was incubated at 36°C for 13 minutes, and radioactivity in the trichloroacetic acic-insoluble fraction was measured by a liquid scintillation counter The first amino acid residue of the tag-peptide is alanine, of unassigned origin (Figure 1; Tu et al, 1995). It is conceivable that this alanine is derived from that aminoacylated to 10Sa RNA. To study the significance of the alanine-accepting, ability of 10Sa RNA for its activity as an mRNA int vitro, we prepared a 10Sa RNA variant possessing A-U in stead of G-U at the third base-pair of the acceptor stem, which is a major identity determinant of ARNA*® (Figure 1; McClain & Foss, 1988; Hou & Schimmel, 1988). This mutant, which cannot be aminoacylated with alanine (Figure 3(a)), stimu- lated neither alanine nor tyrosine incorporation in the presence of poly (U) (Figure 3(b) and 3(¢)). These results indicate that the function as an ala- nine tRNA is essential for the function as an mRNA, supporting the idea that the first alanine of the tag-peptide is derived from that aminoacylated to 10Sa RNA. Our previous study has shown that B. subtilis 10Sa RNA interacts predominantly with 70 $ ribo- somes, but scarcely with the dissociated ribosomal subunits int vivo (Ushida et al,, 1994). E. coli 10% RNA has also been shown to interact with 70S ribosomes (Komine et al, 1996; Tadaki et al., 1996). We then examined the ribosome-binding property of F. coli 10Sa RNA in an in vitro reaction mixture by sucrose density gradient centrifugation. As shown in Figure 4, a small portion of the wild-type 10Sa RNA appeared in the 70 ribosomal fraction, but the mutant possessing the A-U pair did not bind. This result suggests that aminoacylation with alanine is required for efficient association of 10Sa RNA to the ribosome. The absence of any products other than that of 10Sa RNA in the 70S ribosomal fraction (Figure 4) suggests that the tag-peptidle ad- dition is not a result of pre-translational trans-spli- cing between poly (U) and 10Sa RNA.