1887

Microbiology Society logo

Volume 148, Issue 12

Characterization of ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome Free

Abstract

Ribosome recycling factor (RRF), coded for by the locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles. In a cross-species-complementation experiment, it was shown that the RRF protein lacking five amino acids from its C-terminal end (ΔC5RRF) but not the full-length protein (RRF) complemented for its phenotype. It was also shown that the RFF protein (RRF) did not complement LJ14 for . However, simultaneous expression of elongation factor G (EFG) and RRF from resulted in complementation of LJ14. Here it is shown that unlike ΔC5RRF, an equivalent mutant of RRF lacking six amino acids from its C-terminal end (ΔC6RRF) did not complement LJ14. Surprisingly, ΔC6RRF failed to complement the strain even in the presence of homologous EFG (EFG). The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly. However, ribosome-binding assays showed that the mutant protein was compromised in its binding to ribosomes. It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CD, circular dichroism , EcoEFG, E. coli EFG , EcoRRF, full-length E. coli RRF , EFG, elongation factor G; frr, locus coding for RRF , elongation factor G , ESI-MS, electron spray ionization-mass spectroscopy , frrts, locus coding for temperature-sensitive RRF , MtuEFG, M. tuberculosis EFG , MtuRRF, full-length M. tuberculosis RRF , polysome binding , RFF, ribosome recycling factor , termination complex , Thermus thermophilus , TthRRF, full-length T. thermophilus RRF and tubercle bacilli
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-12-3913
2002-12-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/12/1483913a.html?itemId=/content/journal/micro/10.1099/00221287-148-12-3913&mimeType=html&fmt=ahah

References

  1. Clark-Curtis J. E. 1990; Genome structure of mycobacteria. In Molecular Biology of the Mycobacteria pp 77–95 Edited by McFadden J. London: Academic Press;
     [Google Scholar]
  2. Cole S. T., Brosch R., Parkhill J. 39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
     [Google Scholar]
  3. Fujiwara T., Ito K., Nakayashiki T., Nakamura Y. 1999; Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus : evidence for C-terminal modulator element. FEBS Lett 447:297–302 [CrossRef]
     [Google Scholar]
  4. Fujiwara T., Ito K., Nakamura Y. 2001; Functional mapping of ribosome-contact sites in the ribosome recycling factor: a structural view from a tRNA mimic. RNA 7:64–70 [CrossRef]
     [Google Scholar]
  5. Gay N. J. 1984; Construction and characterization of an Escherichia coli strain with a uncI mutation. J Bacteriol 158:820–825
     [Google Scholar]
  6. Girbes T., Cabrer B., Modolell J. 1979; Preparation and assay of purified Escherichia coli polysomes devoid of free ribosomal subunits and endogenous GTPase activities. Methods Enzymol 59:353–362
     [Google Scholar]
  7. Hershey J. W. B. others 1987 In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology pp 613–647 Edited by Neidhardt F. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  8. Inokuchi Y., Hirashima A., Sekine Y., Janosi L., Kaji A. 2000; Role of ribosome recycling factor (RRF) in translational coupling. EMBO J 19:3788–3798 [CrossRef]
     [Google Scholar]
  9. Janosi L., Shimizu I., Kaji A. 1994; Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. Proc Natl Acad Sci USA 91:4249–4253 [CrossRef]
     [Google Scholar]
  10. Janosi L., Hara H., Zhang S., Kaji A. 1996; Ribosome recycling by ribosome recycling factor (RRF) – an important but overlooked step of protein biosynthesis. Adv Biophys 32:121–201 [CrossRef]
     [Google Scholar]
  11. Janosi L., Mottagui-Tabar S., Isaksson L. A. 7 other authors 1998; Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis. EMBO J 17:1141–1151 [CrossRef]
     [Google Scholar]
  12. Janosi L., Mori H., Sekine Y., Abragan J., Janosi R., Hirokawa G., Kaji A. 2000; Mutations influencing the frr gene coding for ribosome recycling factor (RRF). J Mol Biol 295:815–829 [CrossRef]
     [Google Scholar]
  13. Kaji A., Teyssier E., Hirokawa G. 1998; Disassembly of the post-termination complex and reduction of translational error by ribosome recycling factor (RRF) – a possible new target for antibacterial agents. Biochem Biophys Res Commun 250:1–4 [CrossRef]
     [Google Scholar]
  14. Karimi R., Pavlov M. Y., Buckingham R. H., Ehrenberg M. 1999; Novel roles for classical factors at the interface between translation termination and initiation. Mol Cell 3:601–609 [CrossRef]
     [Google Scholar]
  15. Kaufmann S. H., van Embden J. D. 1993; Tuberculosis: a neglected disease strikes back. Trends Microbiol 1:2–5 [CrossRef]
     [Google Scholar]
  16. Kim K. K., Min K., Suh S. W. 2000; Crystal structure of the ribosome recycling factor from Escherichia coli . EMBO J 19:2362–2370 [CrossRef]
     [Google Scholar]
  17. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
     [Google Scholar]
  18. Lowe A. M., Beattie D. T., Deresiewicz R. L. 1998; Identification of novel staphylococcal virulence genes by in vivo expression technology. Mol Microbiol 27:967–976 [CrossRef]
     [Google Scholar]
  19. Mangroo D., Limbach P. A., McCloskey J. A., RajBhandary U. L. 1995; An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation. J Bacteriol 177:2858–2862
     [Google Scholar]
  20. McFadden J. 1996; Recombination in mycobacteria. Mol Microbiol 21:205–211 [CrossRef]
     [Google Scholar]
  21. Ohnishi M., Janosi L., Shuda M., Matsumoto H., Hayashi T., Terawaki Y., Kaji A. 1999; Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor. J Bacteriol 181:1281–1291
     [Google Scholar]
  22. Rao A. R., Varshney U. 2001; Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli . EMBO J 20:2977–2986 [CrossRef]
     [Google Scholar]
  23. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  24. Selmer M., Al-Karadaghi S., Hirokawa G., Kaji A., Liljas A. 1999; Crystal structure of Thermotoga maritima ribosome recycling factor: a tRNA mimic. Science 286:2349–2352 [CrossRef]
     [Google Scholar]
  25. Studier F. W., Moffatt B. A. 1986; Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130 [CrossRef]
     [Google Scholar]
  26. Tai P. C., Davis B. D. 1979; Isolation of polysomes free of initiation factors. Methods Enzymol 59:362–371
     [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354 [CrossRef]
     [Google Scholar]
  28. Toyoda T., Tin O. F., Ito K., Fujiwara T., Kumasaka T., Yamamoto M., Garber M. B., Nakamura Y. 2000; Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch. RNA 6:1432–1444 [CrossRef]
     [Google Scholar]
  29. Vizcaino N., Cloeckaert A., Dubray G., Zygmunt M. S. 1996; Cloning, nucleotide sequence, and expression of the gene coding for a ribosome recycling factor-homologous protein of Brucella melitensis . Infect Immun 64:4834–4837
     [Google Scholar]
  30. Yoshida T., Uchiyama S., Nakano H. 11 other authors 2001; Solution structure of the ribosome recycling factor from Aquifex aeolicus . Biochemistry 40:2387–2396 [CrossRef]
     [Google Scholar]
  31. Young D. B., Cole S. T. 1993; Leprosy, tuberculosis, and the new genetics. J Bacteriol 175:1–6
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-12-3913
Loading
Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome
Microbiology 148, 3913 (2002); https://doi.org/10.1099/00221287-148-12-3913
/content/journal/micro/10.1099/00221287-148-12-3913
/content/journal/micro/10.1099/00221287-148-12-3913
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error