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transfer-messenger RNA

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tmRNA (transfer-messenger RNA, also known as 10Sa RNA and by its genetic name SsrA) is a bacterial RNA molecule with dual tRNA-like and messenger RNA-like properties. The tmRNA forms a ribonucleoprotein complex (tmRNP) together with Small Protein B (SmpB), Elongation Factor Tu (EF-Tu), and ribosomal protein S1. In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein biosynthesis, for example when reaching the end of a messenger RNA which has lost its stop codon. The tmRNA is remarkably versatile: it recycles the stalled ribosome, adds a proteolysis-inducing tag to the unfinished polypeptide, and facilitates the degradation of the aberrant messenger RNA. In the majority of bacteria these functions are carried out by standard one-piece tmRNAs. In other bacterial species, a permuted ssrA gene produces a two-piece tmRNA in which two separate RNA chains are joined by base-pairing.

tmRNA combines features of tRNA and mRNA.

Discovery of tmRNA and Early Work

tmRNA was first designated 10Sa RNA after its resolution in a mixed “10S” electrophoretic fraction of Escherichia coli RNA from the similarly-sized RNase P RNA (10Sb).[1] The presence of pseudouridine in the mixed 10S RNA hinted that tmRNA has modified bases found also in tRNA. The similarity at the 3' end of tmRNA to the T stem-loop of tRNA was first recognized upon sequencing ssrA from Mycobacterium tuberculosis.[2] Subsequent sequence comparison revealed the full tRNA-like domain (TLD) formed by the 5' and 3' ends of tmRNA, including the acceptor stem with elements like those in alanine tRNA that promote its aminoacylation by alanine-tRNA ligase[3]. It also revealed differences from tRNA: the anticodon arm is missing in tmRNA, and the D arm region is a loop without base pairs.

tmRNA Structure

Cartoon ribbon structure of the tRNA-like domain of tmRNA. The domain consists of the 3' and 5' ends of the tmRNA. Image was created using Pymol molecular imaging software for students and data obtained from the RCSB Protein Data Bank file for structure 1J1H[4]

Secondary Structure of the Standard One-Piece tmRNAs

Secondary structure of E. coli tmRNA. Shown are the 5' and 3' ends of the 363-nucleotide RNA chain numbered in increments of ten. Short lines indicate Watson-Crick pairings (G-C and A-U); dots are G-U pairings. Prominent are the tRNA-like domain (TLD), the messenger RNA-like region (MLR), and the four pseudoknots (pk1 to pk4). The MLR encodes the tag peptide between resume and stop codons. RNA helices (numbered one to 12) and their sections (letters) are gray.

The complete E. coli tmRNA secondary structure was elucidated by comparative sequence analysis and structural probing.[5][6] Watson-Crick and G-U base pairs were identified by comparing the bacterial tmRNA sequences using automated computational methods in combination with manual alignment procedures.[7][8] The accompanying figure shows the base pairing pattern of this prototypical tmRNA, which is organized into 12 phylogenetically supported helices (also called pairings P1 to P12), some divided into helical segments.

A prominent feature of every tmRNA is the conserved tRNA-like domain (TLD), composed of helices 1, 12, and 2a (analogs of the tRNA acceptor stem, T-stem and variable stem, respectively), and containing the 5' monophosphate and alanylatable 3' CCA ends. The mRNA-like region (MLR) is in standard tmRNA a large loop containing pseudoknots and a coding sequence (CDS) for the tag peptide, marked by the resume codon and the stop codon. The encoded tag peptide (ANDENYALAA in E. coli) varies among bacteria, perhaps depending on the set of proteases and adaptors available[9].

tmRNAs typically contain four pseudoknots, one (pk1) upstream of the tag peptide CDS, and the other three pseudoknots (pk2 to pk4) downstream of the CDS. The pseudoknot regions, although generally conserved, are evolutionarily plasic. For example, in the (one-piece) tmRNAs of cyanobacteria, pk4 is substituted with two tandemly arranged smaller pseudoknots. This suggests that tmRNA folding outside the TLD can be important, yet the pseudoknot region lacks conserved residues and pseudoknots are among the first structures to be lost as ssrA sequences diverge in plastid and endosymbiont lineages. Base pairing in the three-pseudoknot region of E. coli tmRNA is disrupted during trans-translation.[10][11]

Two-piece tmRNAs

Circularly permuted ssrA has been reported in three major lineages: i) all alphaproteobacteria and the primitive mitochondria of jakobid protists, ii) two disjoint groups of cyanobacteria (Gloeobacter and a clade containing Prochlorococcus and many Synechococcus), and iii) some members of the betaproteobacteria (Cupriavidus and some Rhodocyclales).[12][13] All produce the same overall two-piece (acceptor and coding pieces) form, equivalent to the standard form nicked downstream of the reading frame. None retain more than two pseudoknots compared to the four (or more) of standard tmRNA.

Alphaproteobacteria have two signature sequences: replacement of the typical T-loop sequence TΨCRANY with GGCRGUA, and the sequence AACAGAA in the large loop of the 3´-terminal pseudoknot. In mitochondria, the MLR has been lost, and a remarkable re-permutation of mitochondrial ssrA results in a small one-piece product in Jakoba libera.[14]

The cyanobacteria provide the most plausible case for evolution of a permuted gene from a standard gene, due to remarkable sequence similarities between the two gene types as they occur in different Synechococcus strains.

tmRNA Processing

Most tmRNAs are transcribed as larger precursors which are processed much like tRNA. Cleavage at the 5´ end is by ribonuclease P.[15] Multiple exonucleases can participate in the processing of the 3´ end of tmRNA, although RNase T and RNase PH are most effective.[16][17] Depending on the bacterial species, the 3'-CCA is either encoded or added by tRNA nucleotidyltransferase.

Similar processing at internal sites of permuted precursor tmRNA explains its physical splitting into two pieces. The two-piece tmRNAs have two additional ends whose processing must be considered. For alphaproteobacteria, one 5´ end is the unprocessed start site of transcription.[18] The far 3´ end may in some cases be the result of rho-independent termination.

Three-dimensional Structures

High-resolution structures of the complete tmRNA molecules are currently unavailable and may be difficult to obtain due the inherent flexibility of the MLR. In 2007, the crystal structure of the Thermus thermophilus TLD bound to the SmpB protein was obtained at 3 Å resolution. This structure shows that SmpB mimics the D stem and the anticodon of a canonical tRNA whereas helical section 2a of tmRNA corresponds to the variable arm of tRNA.[19]

A cryo-electron microscopy study of tmRNA at an early stage of trans-translation shows the spatial relationship between the ribosome and the tmRNP (tmRNA bound to the EF-Tu protein). The TLD is located near the GTPase-associated center in the 50S ribosomal subunit; helix 5 and pseudoknots pk2 to pk4 form an arc around the beak of the 30S ribosomal subunit.[20]

tmRNA Mechanism of Action

Cartoon ribbon structure of the tmRNA dedicated binding protein, SmpB. Image was created using Pymol molecular imaging software for students and data obtained from the RCSB Protein Data Bank file for structure 1CZJ[21]

The generally accepted mechanism of action[22][23][24] involves an alanine-charged tmRNA binding the A-site of a stalled ribosome. Upon binding, peptidyl-transfer occurs extending the nascent peptide with the alanine of the charged tmRNA; thus the peptide is moved onto the tmRNA. The TLD is then translocates to the ribosomal P-site. Trans-translocation occurs as the defective mRNA transcript is released and replaced by the ORF of the tmRNA. As the ribosome moves along each codon of the ORF it translates a peptide tag onto the C-terminus[24] of the nascent polypeptide. As mentioned above, the tag contains recognition sites for various ATP-dependent intercellular proteases[25][26]. Translation is terminated as the stop codon within the ORF is recognized and the tagged nascent peptide is released from the ribosome. Once the tag is recognized by cellular proteases the nascent polypeptide is rapidly degraded[25] and the tmRNA then, through sequence specific determinants within the ORF, facilitates the degradation of the truncated mRNA transcript[27]. This model requires, in addition to general translation factors, small protein B (SmpB), elongation factor Tu (EF-Tu), and ribosomal protein S1. SmpB and EF-Tu bind the TLD of the tmRNA and are required for interaction with target ribosomes and the efficient aminoacylation by alanyl-tRNA. The ribosomal protein S1 binds the MLD and is thought to facilitate entry of the ORF into the stalled ribosome[28].

In 2004, Hallier et al.[28] presented results that suggested an alternative mechanism of tmRNA recruitment. In this mechanism SmpB binds the 70S ribosome independently of tmRNA. They propose this binding triggers the initiation of trans-translation by recruiting an SmpB-free alanyl-tmRNA in complex with EF-Tu. Their report does, however, continue to suggest that SmpB is essential for trans-translation initiation.

References

  1. ^ Ray, B. K., & Apirion, D. (1979). Characterization of 10S RNA: a new stable RNA molecule from Escherichia coli. Mol Gen Genet, 174(1), 25-32.
  2. ^ Tyagi JS, and Kinger AK (1992). Identification of the 10Sa RNA structural gene of Mycobacterium tuberculosis. Nucleic Acids Res, 20, 138.
  3. ^ Komine Y, Kitabatake M, Yokogawa T, Nishikawa K, and Inokuchi H (1994). A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc Natl Acad Sci U S A, 91(20), 9223-27
  4. ^ Someya, T., Nameki, N., Hosoi, H., Suzuki, S., Hatanaka, H., Fujii, M., Terada, T., Shirouzu, M., Inoue, Y., Shibata, T., Kuramitsu, S., Yokoyama, S., Kawai, G. (2003) Solution structure of a tmRNA-binding protein, SmpB, from Thermus thermophilus FEBS Lett. 535: 94-100
  5. ^ Williams KP, Bartel DP. Phylogenetic analysis of tmRNA secondary structure. RNA 1996; 2:1306-10.
  6. ^ Felden B, Himeno H, Muto A, McCutcheon JP, Atkins JF, Gesteland RF. Probing the structure of the Escherichia coli 10Sa RNA (tmRNA). RNA 1997; 3:89-103.
  7. ^ Zwieb C, Wower I, Wower J. Comparative sequence analysis of tmRNA. Nucleic Acids Research 1999; 27:2063-71
  8. ^ Andersen ES, Lind-Thomsen A, Knudsen B, Kristensen SE, Havgaard JH, Torarinsson E, Larsen N, Zwieb C, Sestoft P, Kjems J. and Gorodkin, J (2007). Semiautomated improvement of RNA alignments. RNA. 13, 1850-1859.
  9. ^ Gur E, Sauer RT, 2008. Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease. PNAS 105(42):16113-8.
  10. ^ Zwieb C, Wower I, Wower J. Comparative sequence analysis of tmRNA. Nucleic Acids Research 1999; 27:2063-71
  11. ^ Wower IK, Zwieb C, and Wower J. (2005). Transfer-messenger RNA unfolds as it transits the ribosome. RNA, 11, 668-673.
  12. ^ Keiler KC, Shapiro L, Williams KP. tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter. Proc Natl Acad Sci U S A 2000; 97:7778-83.
  13. ^ Sharkady SM, Williams KP. A third lineage with two-piece tmRNA. Nucleic Acids Res 2004; 32:4531-8.
  14. ^ Jacob Y, Seif E, Paquet PO, Lang BF. Loss of the mRNA-like region in mitochondrial tmRNAs of jakobids. RNA 2004; 10:605-14.
  15. ^ Komine Y, Kitabatake M, Yokogawa T, Nishikawa K, and Inokuchi H (1994). A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc Natl Acad Sci U S A, 91(20), 9223-27
  16. ^ Srivastava RA., Srivastava N., and Apirion D. (1992). Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates. Int J Biochem. 24, 737-49
  17. ^ Li Z, Pandit S, Deutscher MP. 3' exoribonucleolytic trimming is a common feature of the maturation of small, stable RNAs in Escherichia coli. Proc Natl Acad Sci U S A. 1998 95, 2856-61.
  18. ^ this paper
  19. ^ Bessho Y, Shibata R, Sekine S; et al. (2007). "Structural basis for functional mimicry of long-variable-arm tRNA by transfer-messenger RNA". Proc. Natl. Acad. Sci. U.S.A. 104 (20): 8293–8. doi:10.1073/pnas.0700402104. PMID 17488812. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)
  20. ^ Valle M, Gillet R, Kaur S, Henne A, Ramakrishnan V, and Frank J. (2003). Visualizing tmRNA entry into a stalled ribosome. Science. 300, 127-130.
  21. ^ Bessho, Y., Shibata, R., Sekine, S., Murayama, K., Higashijima, K., Hori-Takemoto, C., Shirouzu, M., Kuramitsu, S., Yokoyama, S. (2007) Structural basis for functional mimicry of long-variable-arm tRNA by transfer-messenger RNA. Proc.Natl.Acad.Sci.Usa 104: 8293-8298
  22. ^ Cite error: The named reference ref13 was invoked but never defined (see the help page).
  23. ^ Cite error: The named reference ref14 was invoked but never defined (see the help page).
  24. ^ a b Sundermeier, T. R. & Karzai, A. W. Functional SmpB-Ribosome Interactions Require tmRNA. J. Biol. Chem. 282, 34779-34786 (2007).
  25. ^ a b Cite error: The named reference ref1 was invoked but never defined (see the help page).
  26. ^ Cite error: The named reference ref10 was invoked but never defined (see the help page).
  27. ^ Cite error: The named reference ref9 was invoked but never defined (see the help page).
  28. ^ a b Cite error: The named reference ref4 was invoked but never defined (see the help page).

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