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Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: an
M Selvaraj1, A Govindan, A Seshadri
1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
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.
Insights
Mycobacterium tuberculosis ribosome recycling factor (RRF) rigidity hinders its function in E. coli. Disrupting salt bridges increases RRF mobility and partially restores function, offering insights into RRF mechanism.
Area of Science:
- Molecular Biology
- Structural Biology
- Microbiology
Background:
- Ribosome recycling factor (RRF) is crucial for protein synthesis termination.
- Internal domain mobility of RRF is essential for its biological activity.
- Mycobacterium tuberculosis RRF exhibits reduced function in E. coli compared to its native environment.
Purpose of the Study:
- To investigate the structural basis of Mycobacterium tuberculosis RRF's limited complementation activity in E. coli.
- To determine how inter-domain interactions and molecular rigidity affect RRF function.
- To explore the role of specific salt bridges in regulating RRF mobility and activity.
Main Methods:
- Site-directed mutagenesis to disrupt inter-domain salt bridges in M. tuberculosis RRF.
- Complementation assays in E. coli to assess RRF activity with and without co-expressed EF-G.
- X-ray crystallography to determine the structures of wild-type and mutant RRF proteins.
Main Results:
- M. tuberculosis RRF is more rigid than E. coli RRF due to additional salt bridges.
- Disruption of specific salt bridges increased RRF mobility and partially restored its function in E. coli, even without co-expressed EF-G.
- A C-terminal deletion mutant showed reduced activity, potentially due to hinge region rigidification.
Conclusions:
- Inter-domain salt bridges in M. tuberculosis RRF contribute to its structural rigidity and limited functional complementation in E. coli.
- Increased molecular mobility, achieved by disrupting these salt bridges, can partially rescue RRF activity.
- Structural flexibility is a key determinant for RRF function across different bacterial species.
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