Structural organization of Escherichia coli tmRNA

Biochimie (1996) 78, 979-983 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris

Structural organization of Escherichia coli tmRNA B F e l d e n a, H H i m e n o b, A M u t o b, J F A t k i n s c, R F G e s t e l a n d a,c* aHoward Hughes Medical lnstitute ~nd CDepartment of Human Genetics, 6160 Eccles Genetics Bldg, University of Utah, Sah Lake City, Utah 84112, USA: Department of Biology, Faculty of Science, Hirosaki University, Hirosaki 036, Japan

(Received 28 November 1996; accepted 6 December 1996) Summary - - A secondary structure of Evcherichia coli 10Sa RNA (tmRNA) recently proposed on the basis of a variety of chemical and enzymatic probing data combined with phylogenetic analysis (Felden et al, in press), indicates a highly folded structure. Several long-range interactions including pseudoknots are proposed based on comparative analysis of l0 tmRNA genes. Whereas most of the probing data support these predicted secondary structures, several atypical reactivities in specific domains of the molecule suggest structural dynamics, perhaps relating to the complex functions of the molecule as both tRNA and mRNA. The structure of tmRNA has three modular units: a tRNA-like domain, an mRNA-like domain and an intricate connecting unit probably responsible for correct orientation of the two functional parts of the molecule.

tmRNA I structural probing / pseudoknot / covariation Introduction 10Sa RNA or tmRNA is a small stable RNA [1, 2] first found in Escherichia coli and present in many bacteria. tmRNA is encoded by the ssrA gene [3], and disruption of ssrA affects cell growth [4, 5]. tmRNA appears to function both as tRNA and as mRNA. tmRNAs from E coli and Bacillus subtilis have tRNA properties and can be charged in vitro with alanine [5, 6]. The mRNA function is deduced from the recent observation [7] that in E coli the carboxy termini of truncated proteins destined for degradation have identical 1 l-amino acid tags and that the last 10 residues of this peptide tag are encoded by an open reading frame within tmRNA from the E coli ssrA gene. These tags are added to polypeptides translated from mRNAs lacking a termination codon [8], and the added 1 l-amino acid carboxy terminal tag makes the protein a target for specific proteolysis [8]. A model was proposed (fig 1) where charged alanyl-tmRNA, presumably in the ribosomal A site, first donates its alanine to the stalled peptide chain. Then the ribosome transfers decoding to the internal open reading frame in tmRNA to add the encoded 10 amino acid tag sequence [8]. Normal termination at the end ,of the 10 tmRNA codons allows the previously 'trapped' ribosomes to recycle, and the 1 l-amino acid carboxy terminal tag tar-

*Correspondence and reprints

gets the aberrant protein for degradation [8] (for reviews, see [9, 10]). The equivalent model was proposed independently [11] based on in vitro experiments showing tagged extension of polyphenylalanine with polyU as mRNA. This paper summarizes the recent structural data collected on tmRNAs [ 12]. A schematic drawing summarizes the alignment of 10 tmRNA sequences [12], the probing data and phylogenetic analysis of the sequences. The discussion focuses on the structural organization of the molecule in relation to it:, unusual ability to serve both as tRNA and mRNAqike in a trans-translation mechanism where ribosomes cotranslationally switch from one mRNA to another.

Phylogenetic analysis of tmRNAs Sequence comparison of tmRNA genes from nine microorganisms with that of E coli revealed an obvious alignment and enough variants in sequence to look for covariations that would suggest base pairings in a folded structure. Proposed interactions based on these covariations arid on probing data have been reported [12] and are summarized, schematically, in figure 2. The colored boxes denote paired regions with the number of covariations identified for each domain shown in the upper left hand corner and the number of non-canonical pairings that need to be accommodated are shown in the bottom right corner. The domains sort into five helices (HI, H2, H3, H4 and H6) and four pseudoknots (PK1 to PK4). Another helix (H5) and one paired region (R1) are supported less well, requiring several non-canonical pairings.

980

Trans-Translation

ORF

Psits Asite

P site A site

COOH

Tag for Protein Degradation Fig 1. A model for the involvement of E coli tmRNA in tagging truncated proteins by trans-translation.

Helices HI and H6 form the previously proposed tRNA acceptor branch [5, 6] (see the red lines in figure 2) from the two ends of the tmRNA, and are supported by the presence of five covariations for H 1 and 7 for H6 [ 121. A long-range interaction, helix H2 (boxed blue in figure 2), is based on 16 covariations. This is one of the most phylogenetically supported stems of the molecule, but surprisingly some of our probing data argue against it [ 12] (see below). Two other stem loops, H3 and H4, encompassing the coding sequence of tmRNA are proposed on the basis of a few covariations, and are inserted in between two pseudoknots (PKI and PK2). The overall structural organization of tmRNA as depicted in figure 2 emphasizes the compactness and intricate folding of the molecule. The two ends of the molecule are basepaired forming the tRNA-like domain, and the central part of E coli tmRNA is a series of pseudoknots, and two stemloops encompass the coding sequence.

Phylogeny versus probing Covariations identified by sequence comparisons identify potential RNA interactions that must be important for some aspect of function. However, there is no guarantee that the structures predicted by this route will be those that are observed when the molecule is examined in solution. The covariations could represent alternatively folded states that correspond to different steps in a functional pathway that might be influenced by interactions with ribosomes or other cellular components. Some regions of the sequence might be so important that no variation is found but this does not mean that no structure is present. For these reasons it is particularly important to compare covariations with chemical and enzymatic probing data. The probing data based on enzymatic cleavages have allowed discrimination between single- and doublestranded regions within tmRNA, and those obtained from

981

R1

PK1

t

sequence

?K4

tRNA-like domain Fig 2. Schematic representation of the structural organization of closely related tmRNA sequences with E coli based on probing data and phylogeny (data derived from [12]). The color boxes indicate the proposed base-paired domains within the molecule. Within all boxes, numbers at the upper-left comer are the covariations between species. Numbers at the lower-right corners are the mismatches or the putative non-canonical base-pairs. Signs at the lower-left comers indicate if the probing data does (+) or does not (-) match the phylogeny. Both the coding sequence and the tRNA-like domain are indicated. The white boxes are only poorly supported by the phylogney, but are proposed on the basis of the probing data. The white insert within specific boxes (in PK2 and PK3) are internal bulges. The length of the links between the boxes reflects the number of single-stranded nucleotides. The numbering is indicated in red.

chemical modifications have refined the proposed secondary structure [ 12]. The probing data have confirmed the existence in solution of H 1, H3, H4, H5 and H6. H5, however, is only weakly supported by the phylogeny, R I pairing is proposed, but is only partially supported by ooth the prob-

ing and phylogeny. Of the four predicted pseudoknots, three of them (PK 1, PK3 and PK4) are supported by phylogeny and probing. More precisely, the starting and ending nucleotide for each base-paired and single-stranded region have been identified. Moreover, the number of nucleotides cross-

982 ing both the major and minor grooves within the pseudoknots have been determined [ 12]. Some of the probing data argue against phylogenetically supposed, p~red domains in tmRNA [ 12]. This is the case of oneof the two stems of PK2, (PK2.2 according to [12], corresponding to the two orange boxes in figure 2), which is poorly supported by the probing data despite a set of convincing covariations [12]. The stability of this stem is considerably increased in other species (eg three G-C in Haemophilus influenzae, four G-C in Thermus thermophilus), as compared to E coil (only two G-C). H2 is the second ambiguous domain of the molecule. H2 is strongly supported by 16 covariations but is cut by S 1 in both strands [ 12]. Perhaps PK2 and H2 are cases where the covariation and probing analyses reflect alternative structures that are dynamic. For instance, maybe PK2 and/or H2 are not present or are at least unstable on initial contact with the ribosome, but a subsequent rearrangement involves their formation. R I is the third structural domain which is poorly supported by the probing and covariations are also not convinc-

R1

ing. This area of the molecule seems unstable [12], and perhaps several conformations occur in solution.

Structural organization of E coil tmRNA Figure 3 illustrates schematically our current view of the overall structural organization of tmRNAs. The three modular units are proposed to be tightly connected: A tRNA-like domain (in blue), an mRNA-like domain (in red), and a connecting unit that we have termed the 'connector' (in yellow). The aminoacylatable tRNA-like domain is formed by H1 and H6, and mimics a tRNA minihelix containing an acceptor stem (HI) and the analog of a T stem-loop (H6) [5, 6]. This should be sufficient for aminoacylation since a minihelix derived from a tRNAAla can be aminoacylated with alanine [13] and therefore contains the major signals. However, it has been proposed that the tRNA-like domain of tmRNAs could be extended [6, 14]. The structural analog of an anti-

B3

PK1 K2

d o:r.~itj~

H4 PK4 H5

PK2 PK3

Fig 3. Schematic representation of the secondary structure of E coli tmRNA organized into three major modules: a tRNA-like domain in blue, an mRNA-like domain in red linked by a yellow connector. The structural domains (R1, H1 to H6, and PKI to PK4) are similar to that of figure 2 and of [12]. The black filled ovals are the paired regions that are supported by both the phylogeny and probing, whereas the empty ovals are those which are only supported by either the probing or the phylogeny. Note that the location of H5 and its upstream sequence is ambiguous, and that additional data will be required to establish it's precise location within the current model. During its biological role, E coil tmRNA is aminoacylated with alanine, as indicated. The numbering is also shown.

983 codon stem-loop could play an important role, however, during the interaction of tmRNAs with the ribosome. Additional data will be required to test the existence of H5 and to assess its involvement in both aminoacylation and transtranslation functions of E co!i tmRNA. It is tempting to speculate that tmRNA undergoes a conformational change during its switch in roles from a tRNA to an mRNA, and that H5 is only present during the initial contact with the ribosome. The coding sequence is included in a second structural module made of two stem-loops (H3 and H4 in figure 3). It is unclear whether the tmRNA sequences of some of the Gram-positive species (Mycobacterium tuberculosis, B subtilis, Mycoplasma genitalium and Mycoplasma car ricolum) adopt a similar folding of their coding sequence [~ 2]. Strikingly, four stem-loops of E coli tmRNA are located in or near the two functional domains of the molecule, whereas the remaining module is made exclusively of pseudoknots or other long-range interactions. It is perhaps not surprising that the coding sequence is not included in a pseudoknot. However, in the case of aminoacylatable plant viral RNAs, pseudoknots can mimic tRNA acceptor branches (for review see [15]). But in tmRNA, the acceptor stem is made by another long-range interaction involving both ends of the molecule. The third structural unit, the 'connector' encompasses R1, H2, PK1 to PK4 and maybe H5 (fig 3). The tRNA-like and the mRNA-like domains are connected at both ends: R 1 and PK 1 form the first link, and PK2, PK3, PK4 and H5 the second, both connected by H2. The elaborate organization of the connector might allow a tight positioning of the two functional modules during trans-translation. Preliminary mutational analyses of R1 and PKI have shown that their disruptions affect the capacities of translation of E coli tmRNA (Himeno et al, to be published). Future mutational analysis of H2, PK2, PK3 and PK4 will test the structural and functional (aminoacylation and/or translation) importance of the connecting units. Specific deletions of these pseudoknots will help define their contributions in the function of tmRNAs. Much is yet to be learned about the complex role played by tmRNA. How does it fit into the ribosomal A site with its bulky connector and coding sequence? How does it move to the P site and at the same time make its coding sequence accessible to the decoding center? The complex folded state of the molecule in solution must be key and a more detailed analysis by probing, mutations and other structural ap-

proaches is certainly warranted. Early hints of structural dynamics suggest that it will be important to probe tmRNA structure while it is engaged in tile A and P sites.

Acknowledgments RFG is an investigator of the Howard Hughes Medical Institute.

This work was a!so supported by a grant (to JFA) from the US National Institutes of Health (ROI-GM48152) and by a Grant-inAid for the work in Hirosaki from the Ministry of Education, Science and Culture, Japan.

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