An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli. Issue 12 (30th June 2022)

• Record Type: Journal Article
• Authors: Lahry, Kuldeep; Gopal, Aiswarya; Sahu, Amit Kumar; Marbaniang, Carmelita Nora; Shah, Riyaz Ahmad; Mehta, Avani; Varshney, Umesh
• Is Part Of: Journal of molecular biology. Volume 434:Issue 12(2022)
• Publication Date: 2022-06-30
• Subjects: 3GC base pairs -- initiator tRNA -- translation initiation -- RluD -- pseudouridine synthase -- RbfA -- ribosome biogenesis; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2022.167588
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: A point mutation (E265K) in RluD, a large subunit pseudouridine synthase, does not compromise its pseudouridine synthase activity. The E265K mutation reveals a new role of the C-terminal tail domain of RluD in the fidelity of translation initiation. RluD facilitates ribosome biogenesis by contributing to releasing RbfA from the 30S ribosome. The new role of RluD is conserved in both the K- and B-, strains of E. coli . 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.