Abstract
Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.
Similar content being viewed by others
References
Arora A, Chandra NR, Das A, Gopal B, Mande SC, Prakash B, Ramachandran R, Sankaranarayanan R, et al. 2011 Structural biology of Mycobacterium tuberculosis proteins: the Indian efforts. Tuberculosis (Edinb) 91 456–468
Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, et al. 1998 Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54 905–921
Chetnani B, Kumar P, Surolia A and Vijayan M 2010 M. tuberculosis pantothenate kinase: dual substrate specificity and unusual changes in ligand locations. J. Mol. Biol. 400 171–185
Emsley P and Cowtan K 2004 Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60 2126–2132
Fujiwara T, Ito K, Nakayashiki T and 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
Hirashima A and Kaji A 1972 Factor dependent release of ribosomes from messenger RNA. Requirement of two heat stable factors. J. Mol. Biol. 65 43–58
Hirokawa G, Demeshkina N, Iwakura N, Kaji H and Kaji A 2006 The ribosome-recycling step: consensus or controversy? Trends Biochem. Sci. 31 143–149
Janosi L, Mottagui-Tabar S, Isaksson LA, Sekine Y, Ohtsubo E, Zhang S, Goon S, Nelken S, Shuda M and Kaji A 1998 Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis. EMBO J. 17 1141–1151
Kaushal PS, Talawar RK, Krishna PD, Varshney U and Vijayan M 2008 Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D Biol. Crystallogr. 64 551–560
Kim KK, Min K and Suh SW 2000 Crystal structure of the ribosome recycling factor from Escherichia coli. EMBO J. 19 2362–2370
Krishna R, Prabu JR, Manjunath GP, Datta S, Chandra NR, Muniyappa K and Vijayan M 2007 Snapshots of RecA protein involving movement of the C-domain and different conformations of the DNA-binding loops: crystallographic and comparative analysis of 11 structures of Mycobacterium smegmatis RecA. J. Mol. Biol. 367 1130–1144
Nakano H, Uchiyama S, Yoshida T, Ohkubo T, Kato H, Yamagata Y and Kobayashi Y 2002 Crystallization and preliminary X-ray crystallographic studies of a mutant of ribosome recycling factor from Escherichia coli, Arg132Gly. Acta Crystallogr. D Biol. Crystallogr. 58 124–126
Prabu JR, Thamotharan S, Khanduja JS, Chandra NR, Muniyappa K and Vijayan M 2009 Crystallographic and modelling studies on Mycobacterium tuberculosis RuvA Additional role of RuvB-binding domain and inter species variability. Biochim. Biophys. Acta 1794 1001–1009
Rao AR and 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
Schoemaker V and Trueblood KN 1968 On the rigid-body motion of molecules in crystals. Acta Crystallogr. B23 63–76
Rao AR and Varshney U 2002 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 3913–3920
Roy S, Saraswathi R, Chatterji D and Vijayan M 2008 Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function. J. Mol. Biol. 375 948–959
Saikrishnan K, Kalapala SK, Varshney U and Vijayan M 2005 X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications. J. Mol. Biol. 345 29–38
Saikrishnan K, Kalapala SK, Bidya Sagar M, Rao AR, Varshney U and Vijayan M 2004 Purification, crystallization and preliminary X-ray studies of Mycobacterium tuberculosis RRF. Acta Crystallogr. D Biol. Crystallogr. 60 368–370
Selmer M, Al-Karadaghi S, Hirokawa G, Kaji A and Liljas A 1999 Crystal structure of Thermotoga maritima ribosome recycling factor: a tRNA mimic. Science 286 2349–2352
Selvaraj M, Roy S, Singh NS, Sangeetha R, Varshney U and Vijayan M 2007 Structural plasticity and enzyme action: crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase. J. Mol. Biol. 372 186–193
Seshadri A, Singh NS and Varshney U 2010 Recycling of the posttermination complexes of Mycobacterium smegmatis and Escherichia coli ribosomes using heterologous factors. J. Mol. Biol. 401 854–865
Seshadri A and Varshney U 2006 Mechanism of recycling of post-termination ribosomal complexes in eubacteria: a new role of initiation factor 3. J. Biosci. 31 281–289
Storoni LC, McCoy AJ and Read RJ 2004 Likelihood-enhanced fast rotation functions. Acta Crystallogr. D Biol. Crystallogr. 60 432–438
Terwilliger TC, Park MS, Waldo GS, Berendzen J, Hung LW, Kim CY, Smith CV, Sacchettini JC, et al. 2003 The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology. Tuberculosis (Edinb) 83 223–249
Toyoda T, Tin OF, Ito K, Fujiwara T, Kumasaka T, Yamamoto M, Garber MB and 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
Vijayan M 2005 Structural biology of mycobacterial proteins: the Bangalore effort. Tuberculosis (Edinb) 85 357–366
Winn MD, Isupov MN, Murshudov GN 2001 Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57 122–133
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, et al. 2011 Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67 235–242
Acknowledgements
The work has been supported by the Department of Biotechnology, India. X-ray intensity data were collected at the DST supported facility for protein X-ray crystal structure determination and protein design. Part of the computations was carried out at the Interactive Graphics Facility supported by the DBT. MV is the Albert Einstein Professor of the Indian National Science Academy.
Author information
Authors and Affiliations
Corresponding author
Additional information
Corresponding editor: SEYED E HASNAIN
[Selvaraj M, Govindan A, Seshadri A, Dubey B, Varshney U and Vijayan M 2013 Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants. J. Biosci. 38 1–11] DOI 10.1007/s12038-013-9381-0
Rights and permissions
About this article
Cite this article
Selvaraj, M., Govindan, A., Seshadri, A. et al. Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants. J Biosci 38, 845–855 (2013). https://doi.org/10.1007/s12038-013-9381-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12038-013-9381-0