Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 May 15;16(10):2968–2974. doi: 10.1093/emboj/16.10.2968

Rescuing an essential enzyme-RNA complex with a non-essential appended domain.

E F Whelihan 1, P Schimmel 1
PMCID: PMC1169904  PMID: 9184240

Abstract

Certain protein-RNA complexes, such as synthetase-tRNA complexes, are essential for cell survival. These complexes are formed with a precise molecular fit along the interface of the reacting partners, and mutational analyses have shown that amino acid or nucleotide substitutions at the interface can be used to disrupt functional or repair non-functional complexes. In contrast, we demonstrate here a feature of a eukaryote system that rescues a disrupted complex without directly re-engineering the interface. The monomeric yeast Saccharomyces cerevisiae glutaminyl-tRNA synthetase, like several other class I eukaryote tRNA synthetases, has an active-site-containing 'body' that is closely homologous to its Escherichia coli relative, but is tagged at its N-terminus with a novel and dispensable appended domain whose role has been obscure. Because of differences between the yeast and E. coli glutamine tRNAs that presumably perturb the enzyme-tRNA interface, E. coli glutaminyl-tRNA synthetase does not charge yeast tRNA. However, linking the novel appended domain of the yeast to the E. coli enzyme enabled the E. coli protein to function as a yeast enzyme, in vitro and in vivo. The appended domain appears to contribute an RNA interaction that compensates for weak or poor complex formation. In eukaryotes, extra appended domains occur frequently in these proteins. These domains may be essential when there are conditions that would otherwise weaken or disrupt formation of a critical RNA-protein complex. They may also be adapted for other, specialized RNA-related functions in specific instances.

Full Text

The Full Text of this article is available as a PDF (201.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barbarese E., Koppel D. E., Deutscher M. P., Smith C. L., Ainger K., Morgan F., Carson J. H. Protein translation components are colocalized in granules in oligodendrocytes. J Cell Sci. 1995 Aug;108(Pt 8):2781–2790. doi: 10.1242/jcs.108.8.2781. [DOI] [PubMed] [Google Scholar]
  2. Bedouelle H., Guez V., Vidal-Cros A., Hermann M. Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis. J Bacteriol. 1990 Jul;172(7):3940–3945. doi: 10.1128/jb.172.7.3940-3945.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cherniack A. D., Garriga G., Kittle J. D., Jr, Akins R. A., Lambowitz A. M. Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases. Cell. 1990 Aug 24;62(4):745–755. doi: 10.1016/0092-8674(90)90119-y. [DOI] [PubMed] [Google Scholar]
  4. Crothers D. M., Seno T., Söll G. Is there a discriminator site in transfer RNA? Proc Natl Acad Sci U S A. 1972 Oct;69(10):3063–3067. doi: 10.1073/pnas.69.10.3063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eriani G., Delarue M., Poch O., Gangloff J., Moras D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature. 1990 Sep 13;347(6289):203–206. doi: 10.1038/347203a0. [DOI] [PubMed] [Google Scholar]
  6. Harris C. L., Kolanko C. J. Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae. Biochem J. 1995 Jul 1;309(Pt 1):321–324. doi: 10.1042/bj3090321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Inokuchi H., Hoben P., Yamao F., Ozeki H., Söll D. Transfer RNA mischarging mediated by a mutant Escherichia coli glutaminyl-tRNA synthetase. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5076–5080. doi: 10.1073/pnas.81.16.5076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kerjan P., Cerini C., Sémériva M., Mirande M. The multienzyme complex containing nine aminoacyl-tRNA synthetases is ubiquitous from Drosophila to mammals. Biochim Biophys Acta. 1994 Apr 21;1199(3):293–297. doi: 10.1016/0304-4165(94)90009-4. [DOI] [PubMed] [Google Scholar]
  9. Kim S. H., Suddath F. L., Quigley G. J., McPherson A., Sussman J. L., Wang A. H., Seeman N. C., Rich A. Three-dimensional tertiary structure of yeast phenylalanine transfer RNA. Science. 1974 Aug 2;185(4149):435–440. doi: 10.1126/science.185.4149.435. [DOI] [PubMed] [Google Scholar]
  10. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lamour V., Quevillon S., Diriong S., N'Guyen V. C., Lipinski M., Mirande M. Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8670–8674. doi: 10.1073/pnas.91.18.8670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ludmerer S. W., Schimmel P. Construction and analysis of deletions in the amino-terminal extension of glutamine tRNA synthetase of Saccharomyces cerevisiae. J Biol Chem. 1987 Aug 5;262(22):10807–10813. [PubMed] [Google Scholar]
  13. Ludmerer S. W., Schimmel P. Gene for yeast glutamine tRNA synthetase encodes a large amino-terminal extension and provides a strong confirmation of the signature sequence for a group of the aminoacyl-tRNA synthetases. J Biol Chem. 1987 Aug 5;262(22):10801–10806. [PubMed] [Google Scholar]
  14. Ludmerer S. W., Wright D. J., Schimmel P. Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae. A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain. J Biol Chem. 1993 Mar 15;268(8):5519–5523. [PubMed] [Google Scholar]
  15. Mirande M. Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol. 1991;40:95–142. doi: 10.1016/s0079-6603(08)60840-5. [DOI] [PubMed] [Google Scholar]
  16. Mohr G., Caprara M. G., Guo Q., Lambowitz A. M. A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme. Nature. 1994 Jul 14;370(6485):147–150. doi: 10.1038/370147a0. [DOI] [PubMed] [Google Scholar]
  17. Nureki O., Vassylyev D. G., Katayanagi K., Shimizu T., Sekine S., Kigawa T., Miyazawa T., Yokoyama S., Morikawa K. Architectures of class-defining and specific domains of glutamyl-tRNA synthetase. Science. 1995 Mar 31;267(5206):1958–1965. doi: 10.1126/science.7701318. [DOI] [PubMed] [Google Scholar]
  18. Park S. J., Hou Y. M., Schimmel P. A single base pair affects binding and catalytic parameters in the molecular recognition of a transfer RNA. Biochemistry. 1989 Mar 21;28(6):2740–2746. doi: 10.1021/bi00432a056. [DOI] [PubMed] [Google Scholar]
  19. Rho S. B., Lee K. H., Kim J. W., Shiba K., Jo Y. J., Kim S. Interaction between human tRNA synthetases involves repeated sequence elements. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10128–10133. doi: 10.1073/pnas.93.19.10128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rould M. A., Perona J. J., Söll D., Steitz T. A. Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution. Science. 1989 Dec 1;246(4934):1135–1142. doi: 10.1126/science.2479982. [DOI] [PubMed] [Google Scholar]
  21. Schimmel P., Ribas de Pouplana L. Transfer RNA: from minihelix to genetic code. Cell. 1995 Jun 30;81(7):983–986. doi: 10.1016/s0092-8674(05)80002-9. [DOI] [PubMed] [Google Scholar]
  22. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  24. Walter P., Weygand-Durasevic I., Sanni A., Ebel J. P., Fasiolo F. Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme. J Biol Chem. 1989 Oct 15;264(29):17126–17130. [PubMed] [Google Scholar]
  25. Weiss W. A., Friedberg E. C. Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene. J Mol Biol. 1986 Dec 20;192(4):725–735. doi: 10.1016/0022-2836(86)90024-0. [DOI] [PubMed] [Google Scholar]
  26. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES