An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli |
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| Institution: | 1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India;2. Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India;1. Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Be’er Sheva, Israel;2. The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva, Israel;1. Program in Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada;2. Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada;3. SPARC BioCentre, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada;4. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada;5. Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada;6. Departments of Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5G 1L7, Canada;1. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK;2. Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, United States;3. Department of Mathematics, University of York, York, YO10 5DD, UK;4. York Cross-disciplinary Centre for Systems Analysis, University of York, York, YO10 5GE, UK;5. Department of Biology, University of York, York, YO10 5DD, UK;1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi Key Laboratory for Sugarcane Biology, Guangxi University, Nanning 530004, PR China;2. Department of Hepatobiliary Surgery and Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, PR China;1. Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel;2. Department of Plant Molecular Biology, Lausanne University, 1015 Lausanne, Switzerland |
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| Abstract: | The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site. |
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| Keywords: | 3GC base pairs initiator tRNA translation initiation RluD pseudouridine synthase RbfA ribosome biogenesis |
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